Post translational modifications in tuberculosis: ubiquitination paradox

ABSTRACT

Innate immune signaling and xenophagy are crucial innate defense strategies exploited by the host to counteract intracellular pathogens with ubiquitination as a critical regulator of these processes. These pathogens, including Mycobacterium tuberculosis (M. tb), co-opt the host ubiquitin machinery by utilizing secreted or cell surface effectors to dampen innate host defenses. Inversely, the host utilizes ubiquitin ligase-mediated ubiquitination of intracellular pathogens and recruits autophagy receptors to induce xenophagy. In the current article, we discuss the co-option of the ubiquitin pathway by the M. tb virulence effectors.

Abbreviations: ANAPC2: anaphase promoting complex subunit 2; IL: interleukin; Lys: lysine (K); MAPK: mitogen-activated protein kinase; MAP3K7/TAK1; mitogen-activated protein kinase kinase kinase 7; M. tb: Mycobacterium tuberculosis; NFKB/NF-κB: nuclear factor kappa B subunit; PtpA: protein tyrosine phosphatase; SQSTM1/p62: sequestosome 1; V-ATPase: vacuolar-type H+-ATPase; UBA: a eukaryotic-like ubiquitin-associated domain

Mycobacterium tuberculosis (M. tb) is a chronic human pathogen and remains the top infectious killer world-wide. In 2018 alone, M. tb infection was responsible for 10 million new tuberculosis (TB) cases and 1.5 million deaths, despite being a curable disease [1,2]. An estimated one-third of the world’s population is infected with M. tb that acts as a reservoir for future transmissions necessitating development of preventive treatments [3]. Emergence of drug resistance poses a threat for achieving the goal of eradication of TB by 2035 (End TB strategy) due to limited treatment options. Approximately 2/3^rd of the patients with multidrug-resistant tuberculosis could not be successfully treated in 2018 [4]. The untreated cases fuel a global pandemic with strains emerging that are resistant to almost all classes of drugs [5,6]. Thus, there is an urgent need to identify novel therapeutic targets for developing drugs [7] as well as novel vaccine candidates [8] with emphasis on proper route of delivery [9] to tackle this sly pathogen [10].

The pathogenic success of obligate intracellular microbe M. tb pivots on its ability to avoid host-mediated killing and to replicate inside macrophages. Understanding the immune subversion mechanisms deployed by M. tb, while confined inside the phagosomal lumen or macrophage cytosol is necessary for developing effective and selective M. tb-host interface targeted therapeutics. M. tb induced macrophage subversion mechanisms include exploitation of highly conserved cellular pathways including Macroautophagy/autophagy and post-translational modifications such as ubiquitination [11]. Although the activity of ubiquitin in protein turnover, signaling cascades and vesicular trafficking is well documented, the role of ubiquitin in host defense against invading bacterial pathogens is only beginning to be uncovered. Co-evolution of M. tb with the human host has equipped the microbe with effector mechanisms to counter and exploit ubiquitin-dependent processes. In eukaryotes, the selective elimination of invading pathogens or xenophagy requires association of ubiquitin with intracellular bacteria and different autophagic receptor proteins such as SQSTM1/p62, CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) and OPTN (optineurin) which perform the dual role in recognizing ubiquitinated cytosolic bacteria and bringing them into autophagosomes [12]. Ubiquitination of a substrate involves an ATP-dependent binding of ubiquitin through a ubiquitin-activating enzyme (E1), transfer of ubiquitin from E1 to an E2 conjugating enzyme and finally involvement of a ubiquitin ligase E3 that brings the substrate and the E2 enzyme into close proximity to allow attachment of ubiquitin to the substrate.

In a recent publication in Nature, the authors present valuable mechanistic insights of a host E3 ubiquitin ligase ANAPC2 (anaphase promoting complex subunit 2) – a core subunit of the anaphase promoting complex/cyclosome-mediated ubiquitination of M. tb secreted effector protein Rv0222 to subdue host immunity [13]. In another classic example of immune subversion mechanism via co-opting host ubiquitin, it was demonstrated that M. tb deploys a secreted tyrosine phosphatase, PtpA, that binds host ubiquitin to suppress innate host defenses [14]. Interestingly, same phenomenon of ubiquitination is exploited by the host to target mycobacteria for xenophagy [15]. Here ubiquitin decorated pathogens, via the use of surface ubiquitin binding protein that harbors a eukaryotic-like ubiquitin-associated domain (UBA), are targeted to phagophores (autophagosome precursors) for degradation with the help of multiple autophagy receptors (Figure 1). These elegant studies demonstrate the paradoxical exploitation of host ubiquitin by M. tb that reveals unexplored molecular events at host-pathogen interface and emphasizes the need to develop novel and rational interventions to tackle this pathogen.

Figure 1.

Exploitation of ubiquitination during host pathogen interaction influences disease outcome. Eukaryotic host cells utilize this evolutionary conserved phenomenon to decorate mycobacterial surfaces for their targeting to autolysosome via a process of selective autophagy called xenophagy. Conversely, pathogens have gained the ability to exploit the process of ubiquitination to activate phosphatases that modulate innate signaling pathways to suppress production of pro-inflammatory cytokines. Intriguingly, Mycobacterium tuberculosis (M. tb) also co-opts ubiquitination to enhance/suppress production of these cytokines. This paradoxical observation is probably an evolutionary adaptation that limits overt disease to maintain a chronic infection. (AP-1: activator protein 1; IRAK: interleukin 1 receptor associated kinase; IKBKG: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma)

Ubiquitination involves the attachment of either a single ubiquitin molecule or multiple ubiquitin adducts to the lysine residue of a protein target [16]. Each ubiquitin molecule contains seven lysine residues and a free N terminus, thus allowing the formation of a variety of ubiquitin linkages to yield structurally diverse signals [17]. Lys11, Lys63 linked chains and N-terminally linked linear chains provide scaffolding for the recruitment and assembly of signaling complexes whereas Lys48 linked chains predominantly target substrate for proteasomal degradation [16]. Lys11 linked chains, the best studied among atypical chain types, are also established as an additional proteasomal degradation signal, especially in cell cycle regulation [18,19]. Inflammatory pathways emanating from TLRs (toll like receptors) use diverse ubiquitin linkages to assemble signaling molecules for activation of NFKB/NF-κB (nuclear factor kappa B subunit), and MAP kinases that culminate into the production of innate immune effectors against the pathogen. This recent paper [13] elegantly demonstrates how an effector protein, Rv0222 secreted by M. tb, hijacks and co-opts host ubiquitin system to manipulate host inflammatory signaling. ANAPC2, a RING domain-harboring host ubiquitin ligase, interacts and conjugates Lys11 linked ubiquitin to Lys76 of Rv0222. The conjugation of ubiquitin adducts to Rv0222 coordinates the recruitment of host tyrosine phosphatases PTPN6/SHP1 (protein tyrosine phosphatase non-receptor type 6) and PTPN11/SHP2 (protein tyrosine phosphatase non-receptor type 11) to the adaptor protein TRAF6 (TNF receptor associated factor 6) and MAP3K7/TAK1 (mitogen-activated protein kinase kinase kinase 7). This inhibits Lys63 linked ubiquitination and activity of TRAF6 and its downstream signaling. This co-option of the host ubiquitin system leads to hijacking of NFKB and MAP kinase signaling networks that are required for the production of anti-M. tb effector cytokines, thereby effectively dampening the host innate immune responses to promote its survival (Figure 1). The specificity of the ANAPC2 and Rv0222 interaction was confirmed, as silencing the ANAPC2 gene in host cells or mutation in the ubiquitination site of Rv0222 compromises the immune inhibitory function of Rv0222 and dampens M. tb virulence in the mouse model of infection. Another secreted M. tb virulence factor PtpA, a tyrosine phosphatase, harbors unique ubiquitin interacting motif-like (UIML) region and binds with ubiquitin via hydrophobic interactions, leading to the enhanced activation of its phosphatase activity. The consequent effect being dephosphorylation of MAPK/JNK and MAPK/p38 MAP kinases, followed by the inhibition of phagosomal acidification and pro-inflammatory cytokine production [14] (Figure 1). PtpA also inhibits ubiquitin binding of adaptor TAB3 (TGF-beta activated kinase 1 (MAP3K7) binding protein 3) by competitive inhibition that results in the inhibition of NFKB activation. This exploitation of tyrosine phosphatases from pathogen as well as host reveals an interesting interplay of enzymes to suppress immune responses. This ingenious exploitation of host ubiquitination machinery by M. tb augments its pathogenic fitness and appears to be a direct outcome of host-pathogen co-evolution [20]. It is indeed intriguing to find the multitude of ways this pathogen outsmarts its host for survival.

There is a growing list of protein effectors that M. tb secretes via its sophisticated ESX type VII secretion systems to counter host immunity [21]. MPT53 (Rv2878c), a secreted, disulfide bond forming protein of M. tb, directly interacts with MAP3K7/TAK1 (upstream common activator of NFKB and MAP kinase) to promote its ubiquitination and activation in a TLR2 (toll like receptor 2) and MYD88 independent manner [22]. Mechanistically, MPT53 induces disulfide bond formation utilizing Cys210 of MAP3K7/TAK1 thereby facilitating its interaction with TRAFs and TAB1. This leads to the activation of downstream signaling and production of inflammatory cytokines IL1B/IL1β, IL6 and IL12 that promote host protection [22]. This counteractive use of M. tb protein to directly activate a signaling cascade intermediate reveals a novel strategy employed by the host to mount antibacterial defense. Exploring this tug-of-war between the host and pathogen will be interesting to better unveil the pathomechanism of TB disease.

Some of the most successful intracellular pathogens such as Salmonella, Shigella and Legionella also co-opt host ubiquitin machinery to undermine host innate defenses [23,24]. Lys63 and Lys48 linked ubiquitin chains are attached to these pathogens or to membranous structures harboring these bacteria by host ubiquitin ligases such as PRKN/Parkin, SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), RNF166 (ring finger protein 166) and LRSAM1 (leucine rich repeat and sterile alpha motif containing 1) [23–26]. The unique ubiquitin coating of these microbes are recognized and facilitated for xenophagy by host autophagy receptors SQSTM1, NBR1, CALCOCO2 (calcium binding and coiled-coil domain 2), OPTN (optineurin) and TAX1BP1 (Tax1 binding protein 1) for targeting to the lysosomes [23–27]. Similarly, UBQLN1 (ubiquilin 1), a host protein that contains a UBA, interacts with various cell surface proteins of M. tb and recruits ubiquitin therein [28]. The autophagy receptors SQSTM1 and NBR1, thereafter induce clearance of ubiquitin decorated M. tb, mediated by selective autophagy dependent on MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) and lysosomes. Recently, it has been demonstrated that M. tb surface protein PE_PGRS29 (Rv1468c) contains eukaryotic like UBA at its N terminus that binds ubiquitin. Thereafter, it recruits autophagy receptors to induce xenophagy of M. tb to prevent excessive bacterial burden and inflammation [15] (Figure 1). Though, the authors attribute it to the pathogenic strategy to promote persistent infection by preventing overt inflammation due to excessive bacterial burden, it could be a host mediated strategy to induce xenophagy as discussed above. It is intriguing that several other intracellular bacteria have evolved mechanisms to inhibit xenophagy. Legionella pneumophilla exploits a type IV secretion system (T4SS) secreted effector, RavZ to dampen xenophagy by cleaving LC3–PE [29]. Salmonella typhimurium T3SS (type III secretion system) translocated effector SopF is also depicted to be a xenophagy inhibitor. Bacteria induced vacuolar damage facilitates the association of V-ATPase with autophagy protein ATG16L1 (autophagy related 16 like 1) activating xenophagy. However, to counter this host mediated innate immunity, SopF employs ADP-ribosylation on ATP6V0C (ATPase H+ transporting V0 subunit c) subunit of V-ATPase to block xenophagy by disrupting the association between V-ATPase and ATG16L1 [30]. These intricate strategies exploited by the pathogens promote bacterial survival during infection. Taken together, these emerging evidences suggest that there is an intricate and dynamic balance between the host and the pathogen. A disease or protection that ensues after initial interactions is an outcome of cross talk of multiple cascades like the process of ubiquitination. This co-option of the host ubiquitin system is an emerging area in infectious diseases and in future more studies are expected to shed light on this under investigated aspect.

Identification of the Rv0222 mediated exploitation of the host ubiquitin system [13] is a significant finding which will encourage further studies toward an improved understanding of the ubiquitin system at the interface of host-M. tb interactions, particularly the unknown roles of host-originated and M. tb-mimicking E3 ubiquitin ligases, deubiquitinases, and ubiquitin receptors. This will significantly advance drug development efforts utilizing biochemical and genetic approaches. It would be indeed interesting to explore the cross talk of ubiquitination and mycobacterial endogenous mechanism of post translational mechanism, i.e., pupylation [31]. Moreover, exploring physiological consequences of heterotypic ubiquitin conjugation [32] by various host E3 ligases or otherwise via hydrophobic conjugation would introduce altogether a new area of research. Many questions remain, but the demonstration of these mechanistic insights represents a significant step forward to unveil versatility of host pathogen interaction and paves the way for developing effective M. tb-host interface based interventions [33] as well as novel host directed therapies [34,35].

Funding Statement

This work was supported by the Department of Biotechnology, Ministry of Science and Technology [BT/PR12817/COE/34/23/2015]; Department of Biotechnology, Ministry of Science and Technology [BT/PR23099/NER/95/632/2017]; Department of Biotechnology, Ministry of Science and Technology [BT/PR23155/NER/95/634/2017]; National Institutes of Health [AI 143610].

Author contributions

MS, NQ, JAS, AKS, WRB, NZE, and SEH have contributed substantially in different ways in the writing of this manuscript. All the authors have reviewed and approved the final manuscript.

Disclosure statement

The authors declare no competing conflict of interest.