Despite the availability of antibiotics to combat Mycobacterium tuberculosis infections, tuberculosis (TB) is one of the leading causes of death due to infectious disease. The World Health Organization estimates that M. tuberculosis infects one-third of the world population, and 9.4 million new cases of TB were reported in 2009 (WHO, 2010). The bacterium's ability to infect and replicate within the host macrophage is key to M. tuberculosis success as a pathogen. It does so by arresting phagosome maturation and preventing fusion of the bacteria-containing vacuole with the lysosome. In resting macrophages, M. tuberculosis resides in a vacuole resembling the early endosome with a pH of 6.4 and markers such as the Rab5 GTPase (Sturgill-Koszycki et al., 1994; Clemens and Horwitz, 1996; Via et al., 1997; Clemens et al., 2000). The pathogen successfully modulates phagosome trafficking to establish a niche in resting macrophages, but immune activation or the induction of autophagy in infected macrophages shifts the balance toward mycobacterial clearance (Schaible et al., 1998; Via et al., 1998; Gutierrez et al., 2004; Alonso et al., 2007). In activated macrophages, M. tuberculosis is exposed to reactive oxygen and reactive nitrogen intermediates (ROI and RNI), and the bacterial compartment fuses with the acidic and hydrolytic lysosome. Recently, we showed that ubiquitin and ubiquitin-derived peptides (Ub-peptides) contribute to the mycobactericidal activity of the lysosome (Alonso et al., 2007; Purdy et al., 2009). In this review we discuss the role of Ub-peptides in lysosomal killing of M. tuberculosis. We will review the trafficking events that result in delivery of M. tuberculosis and ubiquitin to the lysosome, and we describe our current working model for the mycobactericidal activity of Ub-peptides.

The arrest of M. tuberculosis phagosome maturation in resting macrophages is a complex process that depends upon both cell wall constituents and protein effectors (Rohde et al., 2007). The bioactive mycobacterial lipid trehalose 6,6′-dimycolate (TDM) along with the mannosylated lipoglycans lipoarabinomannan (ManLAM) and higher-order phosphatidyl-myo-inositol mannosides (PIMs) play a role. When purified and coated onto inert particles, these surface molecules can prevent lysosomal fusion of the bead-containing phagosome (Fratti et al., 2003; Indrigo et al., 2003; Vergne et al., 2004; Kang et al., 2005; Torrelles et al., 2006). Recent work indicates that recognition of these mannosylated lipoglycans by specific receptors promotes phagosome maturation arrest and likely prevents a pro-inflammatory response (reviewed in Schlesinger et al., 2008). During macrophage infection M. tuberculosis engages the mannose receptor, and blocking bacterial entry via the mannose receptor increases co-localization of the pathogen with lysosomal markers (Kang et al., 2005). Mycobacterial proteins also contribute to phagosome maturation arrest, and bacterial mutants with reduced ability to arrest phagosome maturation in macrophages were identified in genetic screens (Pethe et al., 2004; MacGurn and Cox, 2007). The incomplete elimination of these mutants by cultured macrophages points to the extensive host-resistance repertoire of M. tuberculosis (reviewed recently by Ehrt and Schnappinger, 2009; Stallings and Glickman, 2010).

Recent work has examined the means by which immune activation and the induction of autophagy promote mycobacterial clearance. Activated macrophages are mycobactericidal through both oxidative and non-oxidative mechanisms (Figure 1). IFN-γ activated macrophages express phagocyte NADPH oxidase (NOX2) and inducible nitric oxide synthase (NOS2) that produce ROI and RNI, respectively. Studies in mice indicate that ROI and RNI play a key role in control of M. tuberculosis infection (Chan et al., 1992; MacMicking et al., 1997; Darwin et al., 2003). However, IFN-γ knockout mice are substantially more susceptible to M. tuberculosis infection than Nos2 knockout mice, indicating the presence of an IFN-γ regulated, NO-independent killing mechanism (Cooper et al., 1993, 2000; Flynn et al., 1993). Activated macrophages are also mycobactericidal because of enhanced fusion between the mycobacteria-containing vacuole with the lysosome. The mechanisms underlying delivery of mycobacterium to the lysosome are beginning to be elucidated. Work by MacMicking et al. (2003) demonstrated a role for the IFN-γ and LPS – responsive GTPase Irgm1 (also known as LRG-47) in host control of mycobacterial infections. Irgm^−/− mice are more susceptible than wild type to M. tuberculosis, similar to IFNR^−/− mice. Further investigation of Irgm^−/− macrophages indicated that these cells were defective in phagosome–lysosome fusion. The presence of PI(3,4)P₂ and PI(3,4,5)P₃ on the M. tuberculosis phagosome recruits Irgm1. Upon binding of Irgm1 to these lipid moieties, Irgm1 interacts with trafficking molecules such as Snapin to promote SNARE complex assembly and fusion of the M. tuberculosis phagosome with late endosomal and lysosomal compartments (Tiwari et al., 2009).

Deretic and colleagues demonstrated that IFN-γ activation also induces autophagy and promotes delivery of mycobacteria to the lysosome via autophagosomes. In these experiments, Irgm localized with autophagosomes upon IFN-γ activation. This observation suggests a close relationship between IFN-γ-mediated and autophagic mycobacterial control (Gutierrez et al., 2004). Autophagy facilitates protein and organelle degradation by the lysosome and has recently emerged as a key component of the innate immune response (see Deretic and Levine, 2009 for a comprehensive review). An autophagosome is formed when an isolation membrane expands and engulfs cytoplasmic material and organelles, then fuses to form a morphologically distinct double-membrane vacuole. Autophagosome formation requires two Atg5-dependent conjugation systems that generate Atg5–Atg12, which complexes with Atg16 on the isolation membrane and ligates LC3 (also known as Atg8) to phosphatidylethanolamine (PE) thereby localizing LC3 to the nascent autophagosome. PE-conjugated LC3 (LC3-II) decorates both the inner and outer leaflet of the autophagosome membrane. Proteolysis by Atg4 can cleave LC3-II from the cytoplasmic facing outer leaflet, and LC3-II on the inner leaflet is trafficked to the lysosome and degraded (Kirisako et al., 2000; Tanida et al., 2004). LC3 has been routinely used as an autophagosome-specific marker for microscopy and to quantify autophagic activity biochemically. Since LC3 mediates membrane tethering and hemifusion in vitro and in vivo, it likely plays an integral role in driving autophagosome formation and expansion (Nakatogawa et al., 2007). Once formed, autophagosomes mature into autophagolysosomes via a late endosomal intermediate.

Autophagy contributes to innate immunity by engulfing cytoplasmic or vacuolar pathogens and delivering them to the lysosome for elimination. The importance of autophagy in controlling infections has been illustrated using murine models with tissue-specific autophagy deficiency. Atg5^−/− macrophages are impaired for autophagosome formation and have reduced mycobactericidal activity (Ponpuak et al., 2010). Impaired control of M. bovis BCG in these experiments may be independent of autophagosome formation itself. Zhao et al. showed that in activated Atg5^−/− macrophages the p47 GTPase Irga6 was not recruited to the T. gondii-vacuole as in wild type macrophages. As a result these macrophages failed to clear the parasite, whereas wild type macrophages promote parasite killing (Zhao et al., 2008). Autophagosome-independent functions of the autophagy machinery, including potential roles in recruitment of p47 GTPase members, are a current focus of research in several laboratories.

Autophagy is induced in macrophages by nutrient starvation, pharmacologically via the drug rapamycin, or through immune signaling. Work by Deretic's group showed that IFN-γ activation stimulates autophagy in primary bone marrow-derived macrophages and macrophage-like cell lines (Gutierrez et al., 2004). Subsequently several groups have demonstrated induction of autophagy via stimulation of pattern recognition receptors (PRRs) including Toll like receptors (TLRs) and nucleotide-binding oligomerization domain-containing (NOD) proteins. LPS induces autophagy via TLR4 in a TRIF-dependent manner, and LPS-induced autophagy in RAW264.7 macrophages results in localization of M. tuberculosis in autophagosomes (Sanjuan et al., 2007; Xu et al., 2007). TLR2 signaling also induces autophagy. Moreover LC3 levels were reduced in macrophages from Tlr2^−/− mice, consistent with a potential role for TLR signaling in enhancing autophagy (Sanjuan et al., 2007). The TLR7 ligands ssRNA and imiquimod strongly induced autophagosome formation in RAW 264.7 and bone marrow-derived macrophages in a MyD88-dependent manner (Delgado et al., 2008). While TLR7 is not implicated in mycobacterial infections, the authors showed that artificially stimulating TLR7-mediated autophagy promoted M. bovis BCG clearance in infected macrophages. Signaling through the cytoplasmic PRRs also stimulates autophagy. Activation of the NOD2 receptors by bacterial cell wall constituents induced autophagy in murine macrophages in vitro and in vivo (Travassos et al., 2010). Recently Brooks et al. showed that human macrophages respond to M. tuberculosis in a NOD2-dependent manner, which implicates this cytoplasmic sensor in control of mycobacterial infections. Lack of functional NOD2 increased bacterial survival, but the mechanism of mycobacterial control was not addressed experimentally (Brooks et al., 2010). In light of the above reports, one possible scenario is that NOD2 participates in recruitment of the autophagic machinery to the M. tuberculosis vacuole and autophagic clearance.

Extensive literature demonstrates ROI-regulation of autophagy, and this was recently reviewed elsewhere (Scherz-Shouval and Elazar, 2010). ROI generated by mitochondria upon nutrient starvation and exogenously added hydrogen peroxide both promote autophagy (Scherz-Shouval et al., 2007; Chen et al., 2009). In macrophages, ROI generated by the phagocyte NADPH oxidase NOX2 also induced autophagy and LC3 accumulation at the phagosome. The phagocytosis of IgG beads and zymosan was associated with LC3 accumulation on the phagosome, and this was blocked using the NOX2 inhibitor diphenyleneiodonium chloride (Huang et al., 2009). While the mechanism by which localized ROI activates autophagy is unclear, ROI generation appears to be key for Fcγ – receptor and TLR-mediated autophagic clearance.

Finally, the immune mediator vitamin D promotes mycobactericidal activity in human macrophages, and the mechanisms underlying this control were elucidated recently. Work by Liu et al. (2006, 2007) showed that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-D3) induces the generation of the mycobactericidal cathelicidin-derived peptide LL-37 in human macrophages. Data from Jo and colleagues subsequently demonstrated that 1,25-D3 induced autophagy in a cathelicidin-dependent manner (Yuk et al., 2009). The cathelicidin-derived peptide LL-37 also directly contributes to the mycobactericidal properties of autophagic macrophages and will be discussed further below.

While our understanding of autophagy as an antimicrobial process has expanded dramatically in the last decade, a number of questions remain with regard to how autophagic clearance initiates and whether this is a directed process. Work by several investigators suggests a link between the phagocytic and autophagic machinery. LC3 was previously considered a marker exclusively for autophagosomes, but it has recently been associated with phagocytic vacuoles that lacked the defining double-membrane structure of autophagosomes: Sanjuan et al. (2007) reported that LC3 recruitment to the phagosomal membrane upon uptake of zymosan. Proteomic analysis of latex bead phagosomes from RAW264.7 macrophages revealed the presence of LC3, and LC3 levels increased upon starvation-induced autophagy (Shui et al., 2008). Finally, using both immunofluorescence microscopy and biochemical approaches Huang et al. (2009) showed LC3 and Atg12 were recruited to nascent phagosomes during Fcγ-receptor and TLR-mediated phagocytosis. Since Atg12 in complex with Atg5 and Atg16 determine the site of LC3 lipidation (Fujita et al., 2008), it is tempting to speculate that LC3 is directly targeted to phagosomal membranes. Combined, these studies suggest closer examination of LC3-associated phagocytosis and phagosome maturation is warranted. Post-phagocytosis, PRRs may recognize bacterial pathogens and recruit the autophagy machinery to cytoplasmic and vacuolar pathogens. In support of this model, NOD2 co-localized with autophagosome-specific proteins (Travassos et al., 2010). However, it remains to be shown experimentally that NOD2 directly targets intracellular bacteria for clearance by the autophagic pathway.

Autophagic and immune responsive control of mycobacterial infections promotes trafficking of the bacterium to the lysosome, however there are a number of remaining questions regarding the mechanism of mycobacterial killing. We found that solubilized lysosomes isolated from resting bone marrow-derived macrophages are bactericidal toward both M. tuberculosis and M. smegmatis (Alonso et al., 2007). Solubilized lysosomal material was fractionated by HPLC, and a single fraction retained bactericidal activity. Mass spec analysis of this fraction revealed that bactericidal activity is associated with ubiquitin peptides. Full-length purified ubiquitin lacks bactericidal activity, but Ub-peptides obtained from digest of purified ubiquitin with cathepsin proteases or a synthesized peptide Ub2 (STLHLVLRLRGG) are bactericidal against Mycobacteria. We subsequently showed that immunodepletion of ubiquitin from the lysosomal extract with a polyclonal antibody against ubiquitin results in diminished bactericidal activity (Purdy et al., 2009). These data suggest that ubiquitylated proteins are delivered to the lysosomal compartment, where cathepsin proteinases release Ub-peptides that possess antimicrobial activity. To establish a mechanism for bacterial killing by Ub-peptides, it was key that the ubiquitin be localized to the lumen of lysosomes where they would come in direct contact with the bacteria. We performed immunoelectron microscopy and found that ubiquitin localizes in the lumen of electron dense lysosomal compartments. Microscopy of M. tuberculosis-infected macrophages indicated that the induction of autophagy enhances localization of ubiquitin to the lumen of bacteria-containing vacuoles (Alonso et al., 2007). To confirm that autophagy resulted in increased lysosomal ubiquitin, we performed immunoblotting on lysosomal lysates isolated from resting, autophagic, and activated macrophages. Lysates from autophagic and activated macrophages have increased levels of ubiquitin relative to lysates from resting macrophages, and this correlates with increased bactericidal activity of these lysosomal lysates when tested in vitro.

The antimicrobial peptide repertoire of macrophages is not limited to Ub-peptides. Other peptides with demonstrated mycobactericidal activity include hepcidin, cathelicidin, and Fau-derived peptides. Hepcidin is expressed in the RAW264.7 macrophage-like cell line and co-localizes with M. tuberculosis in IFN-γ activated macrophages (Sow et al., 2007). In human-derived macrophages, Vitamin D and TLR2 stimulation induce transcription of cathelicidin. In autophagic macrophages, cathelicidin co-localizes in vacuoles containing M. tuberculosis, and this is associated with decreased bacterial viability (Liu et al., 2006, 2007; Yuk et al., 2009). Deretic and colleagues recently reported the lysosomal generation of Fau-derived antimycobacterial peptides (Ponpuak et al., 2010). Fau is a precursor protein comprised of a 74-amino acid (aa) polypeptide with 38% identity to ubiquitin fused to the 59-aa ribosomal protein S30. The S30 domain, designated ubiquicidin, was identified as a component of IFN-γ stimulated RAW 264.7 macrophages that possess antimicrobial activity against Listeria monocytogenes (Hiemstra et al., 1999). Fau co-localizes with M. bovis BCG phagosomes in autophagic RAW 264.7 macrophages. Digestion of S30/ubiquicidin in vitro with cathepsin L-generated mycobactericidal peptides, and depletion of Fau from phagosomal extracts is associated with reduced bactericidal capacity. Since Fau also contributes mycobactericidal peptides to lysosomal extracts, our data indicating that some bactericidal activity remained upon ubiquitin immunodepletion of lysosomal extracts is not unexpected. In the lysosome, it is likely that Ub-peptides and Fau-derived peptides act synergistically with the complex mixture of hydrolytic enzymes present in the lysosomal lumen, including proteases and lipases, to promote bacterial killing.

As outlined below, our data suggest both the multivesicular body (MVB)/endocytic pathway and autophagy are the source for lysosomal ubiquitin (Figure 2). Ubiquitin is best known as a post-translational addition to proteins targeting them for degradation through the proteasome (Ciechanover, 2009). While polyubiquitylation via Lys48-linked conjugation targets proteins for proteasomal degradation, mono-ubiquitylation, and Lys63-linked polyubiquitylation specifies which proteins should be recycled or sequestered by MVBs and the endocytic pathway (Thrower et al., 2000; Lauwers et al., 2009). The formation of MVBs is important for the recycling and turnover of membrane proteins such as activated growth factor, hormone, and cytokine receptors. MVBs also traffic misfolded membrane proteins for lysosomal degradation and thus contribute to cellular protein quality control. Autophagy also participates in the clearance of ubiquitylated proteins in a proteasome-independent manner. There is some overlap in the two pathways since both MVBs and autophagosomes deliver their contents to the lysosome via a late endosomal intermediate.

We have used biochemical and genetic approaches to better understand the bactericidal activity of lysosomal Ub-peptides and their interactions with mycobacteria. Many of our studies utilize the synthetic model Ub-peptide Ub2, which was originally identified by Kieffer et al. (2003) as an antifungal peptide that also had activity against some Gram-positive bacteria. Our model that Ub-peptides contribute to the mycobactericidal capacity of the lysosome is dependent on the ability of these peptides to function at low pH. Our in vitro experiments with the synthetic bactericidal ubiquitin peptide Ub2 are performed in standard mycobacterial 7H9 media at pH 6.6. Immature and mature mycobacteria-containing phagosomes are at pH 6.2 and pH 5–5.5, respectively (Sturgill-Koszycki et al., 1994; Schaible et al., 1998; Via et al., 1998; de Chastellier and Thilo, 2006). To determine whether pH affected the bactericidal capacity of Ub2, we treated bacteria in 7H9 media at pH 5, pH 5.5, pH 6, and pH 6.6. Ub2 was functional at each of these pH and exhibited the greatest bactericidal activity at pH 5.5 and pH 6 (Purdy et al., 2009). These data demonstrate that Ub-peptides are functional at the physiological relevant pH of the phagolysosomal pathway.

Since Ub2 is a 12-aa positively charged peptide, we predicted that it might behave like a cationic antimicrobial peptide. These conserved components of innate immunity are thought to act by disrupting the bacterial membrane. To directly measure mycobacterial membrane integrity, an assay based upon a pH-sensitive fluorophore was performed on intact bacteria (Figure 3). The cytoplasm of M. smegmatis was labeled with the membrane-permeable probe 5-chloromethylfluorescein diacetate (CMFDA), which is processed into a membrane-impermeable form following uptake. The fluorescence emission of fluorescein at 520 nm is pH-sensitive upon excitation at 490 nm, but is pH-insensitive when excited at 450 nm. The intracellular pH of the labeled bacteria was neutral, and the bacteria were placed in suspension buffered at pH 5.5. The loss of membrane integrity was therefore measured as a shift in fluorescence upon exposure of the intracellular fluorophore to the extracellular pH. As a positive control bacteria were treated for 5 min with the ionophore nigericin. This resulted in a fluorescence emission shift indicating that the intracellular pH had dropped from pH 7 to pH 5.5 (Figure 3). A 5-min treatment with Ub2 resulted in a drop of intracellular pH to pH 5.8. These experiments show that Ub2 treatment impairs membrane function and results in exposure to external pH (Figure 3 and Purdy et al., 2009). Ongoing work in our lab indicates that Ub2 interacts with membranes and adopts a stable structure in membrane mimetic environments. Peptide charge and primary sequence is important for both stable insertion into membranes and bactericidal activity. While there is no significant homology between ubiquitin and the S30/ubiquicidin domain of Fau, both proteins are highly composed of positively charged amino acids. Therefore, the peptides generated from ubiquitin and Fau are likely to be cationic, a property that is likely to be important for their mycobactericidal function. Our data on Ub2 are consistent with the properties of cationic antimicrobial peptides that are thought to mediate bacterial killing through multimerizing and inserting into membranes. Some antimicrobial peptides may also directly inhibit intracellular processes such as nucleic acid or protein synthesis (Brogden, 2005), but we do not have any evidence to suggest that Ub2 functions in this manner.

We hypothesized that isolation of bacterial mutants with increased resistance to the bactericidal ubiquitin-derived peptide Ub2 would provide insight into its mechanism of action. We screened a transposon mutant library for M. smegmatis mutants that exhibited increased resistance to the bactericidal activity of the synthetic ubiquitin-derived peptide Ub2 (Purdy et al., 2009). The Ub2-resistant mutants were more resistant to macrophage killing, consistent with our observation that Ub-peptides contribute to lysosomal killing. A unifying feature of the Ub2-resistant mutants was that they exhibited reduced outer membrane permeability relative to wild type bacteria. To determine whether reduced membrane permeability in Ub2-resistant mutants rendered these bacteria more resistant to other antimicrobial peptides, the M. smegmatis Ub2-resistant mutants were exposed to the cathelicidin LL-37, an LL-37-derived peptide with increased mycobactericidal activity (LL-37 18–37), hepcidin, and granulysin-derived peptide granF2. The four Ub2-resistant M. smegmatis mutants were also resistant to the bactericidal action of these other antimicrobial peptides (Purdy et al., 2009). Our data support a model where the access of host antimicrobial peptides, including Ub-peptides, to the mycobacterial inner membrane depends on outer membrane permeability. Once these peptides gain access to the inner membrane we propose that they multimerize, insert into the membrane and form pores or otherwise disrupt membrane integrity of the bacterium. Membrane damage likely exposes the bacterium to external conditions such as the low pH of the lysosome and promotes cytoplasmic leakage over time that contributes to bacterial death. The contribution of low membrane permeability to the intrinsic resistance of mycobacteria has long been appreciated (Barry, 2001). Our findings emphasize the contribution of this intrinsic resistance to host antimicrobial compounds and the intracellular environment.

Autophagy has emerged as a key component in the host defense against infection by intracellular pathogens. In the context of M. tuberculosis infections, autophagy plays two roles. First, autophagy provides a mechanism by which cytoplasmic material is delivered to the lysosome where digest of some proteins releases mycobactericidal peptides. Second, autophagy promotes fusion of the M. tuberculosis-containing phagosome with the lysosome and thus delivers the pathogen to a compartment enriched with mycobactericidal peptides. Our work is focused on the mycobactericidal properties of Ub-peptides, namely their ability to disrupt mycobacterial membranes. In the lysosome, it is likely that Ub-peptides act synergistically with other mycobactericidal peptides generated in the lysosome and the complex mixture of lysosomal hydrolases to promote bacterial killing. We anticipate that future studies will fully elucidate the Ub-peptide mechanism of action and determine whether this mechanism is shared among lysosomal mycobactericidal peptides.

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.