PUPylation Provides the Punch As Mycobacterium tuberculosis Battles the Host Macrophage

Abstract

The proteasome machinery has been shown to provide Mycobacterium tuberculosis (Mtb) with the ability to protect itself from the damaging effects of reactive nitrogen intermediates. In their recent paper, Darwin and colleagues identify the protein modifier in Mtb that targets substrates for degradation in the Mtb proteasome.

Tuberculosis is primarily acquired through inhalation of airborne droplets containing Mycobacterium tuberculosis (Mtb). Subsequently, the bacteria travel to distal regions of the lung and are engulfed by resident macrophages. Mtb engages several different phagocytic receptors to invade the macrophage, and once inside it successfully evades destruction by the innate microbicidal machinery. Seminal studies performed nearly three decades ago by Armstrong and D’Arcy Hart (Armstrong and Hart, 1975) have shaped our understanding of how Mtb might resist killing inside the macrophage. Their work showed that Mtb vacuoles did not fuse with the lysosomal compartment. Substantive work from independent investigators (Russell, 2007) has expanded on this paradigm to provide a detailed understanding of the molecular events that arrest the maturation of the Mtb phagosome and prevent its normal biogenesis and progression to fusion with the lysosomal compartment. In the battle of the host against pathogen, the first round goes to Mtb.

As the battle progresses, the host counteracts through the activation of adaptive immunity. By inducing immunity-related GTPase Irgm1 and activating the autophagy pathway in infected cells, IFN-γ, a key Th1 effector molecule of the adaptive immune compartment, is able to revoke the restriction on Mtb phagosome maturation and expose Mtb to the antimicrobial contents of the lysosomal compartment (Deretic, 2006). IFN-γ, in conjunction with TNF, upregulates NOS2 and facilitates the production of reactive oxygen and reactive nitrogen intermediates (RNI) within the phagolysosome, resulting in Mtb killing (Nathan and Shiloh, 2000). In vivo, as the battle progresses, an orchestrated series of Th1 dominant adaptive immune pathways are activated to culminate in a granuloma at the initial foci of infection (Russell, 2007). At this stage, the mycobacteria are contained within the granuloma with minimal collateral damage to lung tissue. The second round goes to the host macrophage.

A few conniving bacteria, however, escape the damaging effects of RNI and other toxic molecules generated within the phagosomes. These bacteria continue to persist as nonreplicative forms with the ability to reactivate later and continue the cycle of human to human transmission. The third round goes to Mtb.

Clearly, Mtb is equipped with defense mechanisms that can counteract host-induced oxidative and nitrosative stress to ultimately emerge as the winner. Nathan and colleagues identified Mtb genes that provide defense against RNI (Darwin et al., 2003). They screened a library of 10,000 Mtb transposon mutants for NO sensitivity and identified mutants with insertions in proteasome associated genes, mpa and pafA, providing the first clue that the proteasome pathway afforded Mtb a means to protect itself from toxic RNI. The authors also showed that the mpa gene encoded an ATPase that shared homology with ATPases found in the regulatory cap of the eukaryotic 26S proteasome and most likely functioned in channeling proteins to the proteosome core, while the pafA gene was shown to encode for a proteasome accessory factor. Both mpa and pafA mutants of Mtb were attenuated in mice, signifying the importance of the proteasome pathway to Mtb survival in vivo. Furthermore, this study also demonstrated that specific inhibitors of the proteasome prevented Mtb growth and its ability to resist the toxic effects of RNI. Subsequently, Darwin and colleagues (Pearce et al., 2006) identified FabD and PanB, enzymes involved in the biosynthesis of fatty acids and polyketides, as the natural substrates of the Mtb proteasome. Although these studies provided direct evidence that Mpa and PafA, together with the proteasome protease, regulate protein degradation and disposal in Mtb, additional substrate characterization is required to exactly define the mechanism behind the attenuated virulence of the mpa and pafA mutants.

The studies so far establish that the proteasome regulates Mtb growth in vivo; however, the essentiality of the proteasome to Mtb persistence in vivo was validated in a report from Ehrt’s group (Gandotra et al., 2007). The authors demonstrated that conditional silencing of prcBA, genes encoding Mtb’s gated proteasome core, during chronic infection, prevented bacterial persistence. Interestingly, IFN-γ−/− mice infected with proteasome-silenced Mtb had a moderately better survival rate in comparison with mice infected with Mtb containing intact proteasome, suggesting a more multifaceted role for the proteasome in regulating Mtb growth in vivo.

Together, these studies strongly support that Mtb uses its proteasome machinery to adapt to the host microenvironment and achieve its persistent lifestyle. However, as this storyline developed, a piece of information that was still missing was the process that marked Mtb proteins and designated them for degradation in the proteasome. In eukaryotes, proteins selected for proteasome-mediated removal are tagged with polyubiquitin chains for recognition by the proteasome machinery, but genes encoding this protein modifier have not been identified in the Mtb genome. The missing piece to the story was recently provided in elegant studies by Darwin and colleagues (Pearce et al., 2008), in which they identified a prokaryotic ubiquitin-like protein, Pup, as the Mtb equivalent of the eukaryotic protein modifier ubiquitin. The authors coined the term pupylation to describe the process of conjugation of Pup to its proteasome substrates. Using tandem affinity chromatography to purify pupylated substrates and subsequent mass spectrometric characterization of the interaction between Pup and the substrate revealed that pupylation occurred on specific lysine residues of protein substrates prior to degradation, similar to what is seen during ubiquitylation. Absence of pupylated proteins with attendant accumulation of substrates in PafA mutants provided direct proof that pupylated proteins are destined for degradation.

In eukaryotic organisms, targeted elimination of proteins by the ubiquitin-proteasome pathway is crucial to the regulation of several cellular processes. It would be interesting to see if in an analogous manner the Pup-proteasome pathway also has multifaceted functions in Mtb. Furthermore, using rabbit and guinea pig models, it will be important to ascertain whether the proteasomal pathway plays a role in Mtb’s ability to survive within hypoxic granulomas. Can the role of the proteasome be extended to protection from other insults, besides nitrosative, inside the macrophage? It would be worth exploring if Mtb will use a similar mechanism of removal of damaged proteins via the proteasome to resist antimicrobial peptides such as cathelicidins that block Mtb proliferation in human macrophages (Liu et al., 2006).

Notwithstanding these questions, the Mtb proteasome studies indicate that this machinery is not only necessary for Mtb to survive and replicate in the host, but importantly it is also essential for its ability to persist in the host in a nonreplicative state. The identification of Pup as the protein modifier in the Mtb proteasome pathway (Pearce et al., 2008) provides a great means to identify and characterize Mtb substrates targeted for the proteasome. Identification of such substrates will then distinguish Mtb pathways involved in its in vivo survival and form the basis of rational drug design. Indeed, the Mtb proteasome has already proven useful in this regard. Recent work has identified that a certain class of chemical inhibitors called rhodanines can inhibit dihydrolipoamide acyltrasferase, an enzyme required by Mtb to resist the action of RNI (Bryk et al., 2008). Clearly, the Mtb proteasome pathway is an exciting area for discovering new classes of Mtb drug targets. The host might have lost the battle, but it might just survive to win the war!