Autophagy in Immune Defense Against Mycobacterium tuberculosis

[Autophagy 2:3, 175-178, July/August/September 2006]; ©2006 Landes Bioscience

Autophagy in Immune Defense Against Mycobacterium tuberculosis Review: Spotlight on Bacterial Pathogenesis

ABSTRACT

Isabelle Vergne Sudha Singh Esteban Roberts George Kyei Sharon Master James Harris Sergio de Haro John Naylor Alex Davis Monica Delgado Vojo Deretic*

IST

RIB

UT E

.

Autophagy is a newly recognized innate and adaptive immunity defense against intracellular pathogens, in keeping with its role as a cytoplasmic maintenance pathway. Induction of autophagy by physiological, pharmacological or immunological means can eliminate intracellular Mycobacterium tuberculosis, providing one of the first examples of the immunological role of autophagy. Under normal circumstances, M. Tuberculosis survives in macrophages by inhibiting phagolysosome biogenesis. Induction of autophagy overcomes the mycobacterial phagosome maturation block, and delivers the tubercle bacilli to degradative compartments where they are eliminated.

OT D

MYCOBACTERIUM TUBERCULOSIS, MACROPHAGE INFECTION AND SURVIVAL IN A SPECIALIZED PHAGOSOME

ON

M. TUBERCULOSIS BLOCKS PHAGOLYSOSOME BIOGENESIS BY INTERFERING WITH PHOSPHATIDYLINOSITOL 3-PHOSPHATE GENERATION

IEN

Received 04/05/06; Accepted 04/07/06

.D

*Correspondence to: Vojo Deretic; Department of Molecular Genetics & Microbiology; University of New Mexico Health Sciences Center; 915 Camino de Salud, NE; Albuquerque, New Mexico 87131-001 USA; Tel.: 505.272.0291; Fax: 505.272.5309; Email: [email protected]

CE

Departments of Molecular Genetics and Microbiology and Cell Biology and Physiology; University of New Mexico School of Medicine; Albuquerque, New Mexico USA

Tuberculosis is one of the most prevalent infectious diseases worldwide. The etiologic agent, Mycobacterium tuberculosis, infects over a billion people globally, following a cycle of infection, latency, reactivation and transmission. This cycle rests upon mycobacterial entry into the host macrophages where it resides in phagosomes that remain immature and do not acquire phagolysosomal degradative characteristics.1,2 The most distinguishing features of the M. tuberculosis phagosome are its incomplete lumenal acidification and low abundance of lysosomal hydrolases such as cathepsin D.2 Apart from lacking direct bactericidal mechanisms, mycobacterial phagosomes display a limited capacity for antigen processing.3

SC

Previously published online as an Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=2786

It has been known for over a decade that the phagolysosome biogenesis block initiated by M. tuberculosis occurs between the maturation stages controlled by the small GTP binding proteins Rab5 (early endosomal) and Rab7 (late endosomal).4 This has prompted a search for Rab5 effectors missing on the mycobacterial phagosome and has lead to the observation that phosphatidylinositol 3-phosphate (PI3P)-binding effectors EEA1 and Hrs are underrepresented on mycobacterial phagosomes (Fig. 1).5,6 EEA1 and Hrs recognize PI3P via their FYVE domains; EEA1 also interacts with endosomal Rab proteins.7,8 PI3P and the effectors that bind to membranes earmarked by this phosphoinositide are required for phagosome maturation.5,9 PI3P is generated by the type III phosphatidylinositol 3-kinase (PI3K) hVPS34.10 Consequently, these studies have placed hVPS34 and its lipid kinase product, PI3P, at the center of phagolysosome biogenesis and have established that the inhibition of PI3P generation on mycobacteria-containing phagosomes leads to M. tuberculosis phagosome maturation arrest.1,5,6,9,11-14 At least two mycobacterial products prevent generation or maintenance of PI3P on the phagosome; lipoarabinomannan, a highly glycosylated phosphatidylinositol, prevents acquisition of active hVPS34 by the phagosome,15 while SapM, a PI3P phosphatase, hydrolyzes any residual PI3P.13

BIO

KEY WORDS

ND

ACKNOWLEDGEMENTS

ES

autophagosome, phagosome, phosphatidylinositol, interferon, GTpase

©

20

06

LA

This work was supported by NIH grants AI45148 and AI42999.

AUTOPHAGY: A PI3P-DEPENDENT PROCESS THAT CAN OVERCOME MYCOBACTERIAL PHAGOLYSOSOME BIOGENESIS ARREST

The finding that PI3P is a key membrane trafficking regulatory lipid required for phagosomal maturation, has triggered a search for processes that could stimulate the generation of PI3P and/or potentially bypass the mycobacterial inhibition of PI3P production. One of the considered pathways was autophagy. Autophagy is a fundamental www.landesbioscience.com

Autophagy

175

Autophagy in Immune Defense Against Mycobacterium tuberculosis

cellular process that turns over stable macromolecules, e.g., long-lived proteins, at times of nutritional demands, such as starvation or growth factor withdrawal. Autophagy also patrols the cytoplasm and eliminates surplus or damaged organelles. It has been shown that induction of autophagy by pharmacological (rapamycin) or physiological (amino acid starvation) means promotes elimination of intracellular M. tuberculosis in a wortmannin and 3-methyladenine (3-MA)-sensitive manner.16 Another emerging connection between autophagy and immunity is an acknowledged role of autophagy in the endogenous pathway of MHC II cytosolic antigen processing and presentation.17,18 Whether M. tuberculosis antigen processing and presentation are also enhanced upon induction of autophagy, an issue of potential significance for development of improved vaccines, remains to be determined.

BIOACTIVE LIPIDS AND Th2 CYTOKINES AS MODULATORS OF AUTOPHAGY AND THEIR RELATIONSHIP TO M. TUBERCULOSIS

Figure 1. Autophagy: a phosphatidylinositol 3-phosphate (PI3P) dependent cellular process that promotes killing of intracellular M. tuberculosis. Once phagocytosed by macrophages, M. tuberculosis resides and survives in immature phagosomes by blocking phagolysosome biogenesis (right blue panel). The block results from inhibition of PI3P production on the phagosomal membranes. PI3P is a crucial phosphoinositide that allows the recruitment to the phagosome of PI3P-binding proteins, EEA1 and Hrs, the key effectors in phagolysosome biogenesis. Induction of another PI3P-dependent process, autophagy, by physiological (starvation), pharmacological (rapamycin) or immunological (IFN-γ) treatment overcomes the mycobacterial phagolysosome biogenesis block resulting in M. tuberculosis killing (left yellow panel). Atg, autophagy proteins; LC3, mammalian Atg8 homologue required for autophagy.

Several bioactive lipids shown to have anti-mycobacterial activity play a role in autophagy. It has been shown that sphingolipid messengers can induce autophagy. Of those, ceramide19 activates both autophagy and apoptosis, possibly due to a high increase in beclin-1 levels which binds to and soaks up Bcl-2 and thus may cause cell death.20 Sphingosine 1-phosphate (S1P) induces only autophagy and promotes cell survival.21 Vitamin D3 analogs can also induce autophagy.22 While the pathways and mechanisms involved in these processes remain to be delineated, it is of note that S1P,23 ceramide24 and vitamin D325,26 have been implicated as agents controlling intracellular mycobacteria. The mode of action of these compounds is not established at present, but we note that it is possible that there is an autophagic component in their antituberculosis activity. Recently, cholesterol depletion has been reported to promote sequestration of Mycobacterium avium, a facultative intracellular pathogen, into an autolysosome;27 however, this treatment did not lead to killing of the bacteria. One interpretation of these findings is that cholesterol may be required for the establishment of a degradative autolysosome, opening interesting possibilities for probing the lipid composition of autophagic membranes and linking them with functional properties of the autophagosome. The putative hypervirulent strains of M. tuberculosis HN878 and additional W/Beijing isolates induce IL-13 expression, which, along with IL-4, is a key Th2 response cytokine. Th2 cytokines counteract the beneficial immunological effects of Th1 cytokines, e.g., IFN-γ, and are considered to be counterproductive in control of mycobacterial disease.28 Importantly, IL-13 inhibits autophagy by activating the Akt/PKB pathway,29 which is a major activator of Tor and thus inhibits autophagy. IL-13 has already been shown to inhibit 176

autophagy in nonimmune cells, while its potential equivalent role in macrophages remains to be determined.

IFN-γγ MEDIATED AUTOPHAGY AND IMMUNITY RELATED GTPases

Immunological induction of autophagy by IFN-γ,30 a Th1 cytokine strongly correlating with protective immunity against tuberculosis, bypasses phagolysosome biogenesis arrest.16 This provides a link between immunological control of autophagy and control of intracellular pathogens.31,32 Autophagy can kill intracellular mycobacteria,16 thus explaining in part the anti-tuberculosis action of IFN-γ. The stimulation of these processes by IFN-γ depends on the intermediate size (47 kDa) GTPase Irgm1 (also known as LRG-47).16 Irgm1 (LRG-47) is a member of the family of Immunity Related GTPases (IRG),33 inducible by interferon-γ in mice.34 Since Irgm1 (LRG-47) has been shown to play a critical role in the control of M. tuberculosis in murine infection models of tuberculosis,34 this further firms up the connection between IFN-γ and autophagy as a protective immunity mechanism involved in the elimination of the tubercle bacilli. The mouse genome encodes 22 complete Irg genes33 grouped in three clusters on the chromosomes 7, 11 and 18. The previously known murine p47 GTPases, implicated in immunological control of intracellular pathogens, are scattered within these clusters: LRG-47 is Irgm1, TGTP is Irgb6, IGTP is Irgm3, GTPI is Irgm2, and IIGP1 is IrgA6. The human genome has only three IRG genes: IRGC, IRGQ and IRGM33 with IRGM being the candidate human orthologue of the murine Irgm1. It is not known at present whether the human

Autophagy

2006; Vol. 2 Issue 3


gene IRGM plays a role in autophagy by analogy to Irgm1 in murine macrophages. The precise mode of action of IRG proteins is not known. They might act as signaling GTPases, but it is just as possible that they function as mechanoenzymes. IRG dimerize or oligomerize33,35 similarly to dynamin-like GTPases involved in membrane remodeling. The sources of membranes for the formation and elongation of autophagosomes are presently unclear, but they are believed to originate from the ER or a combination of ER, Golgi, and endosomes.36 Eleven Irgs have an N-terminal myristoylation signal MGXXXS suggesting membrane association. Irgm3 (IGTP) localizes to the ER.37 Other uncharacterized motifs within the protein sequence seem to target Irgm1 to membranes.38 Irgm1 (LRG-47) appears to be exclusively membrane associated, localized to the Golgi with the ability to transfer to nascent phagosomes.38 Myristoylation of Irga6 (IIGP1) plays a role in its binding to ER membranes.38 Interestingly, Irga6 interacts with Hook3,39 a member of the family of microtubule binding proteins;40 microtubules play an important role in autophagy. We propose that at least some of IRG factors function in delivery, or of shaping, of membranes during elongation of autophagosomes, enabling these organelles to engulf large objects, such as phagosomes containing clumps of mycobacteria.

CONCLUSION

Autophagy plays a role in the immunological control of intracellular pathogens involving both innate and adaptive immunity mechanisms.31 A term, immunophagy, has been proposed to indicate this specialized function of autophagy (V. Deretic, Curr Opin Immunol, In press). The infection of macrophages by M. tuberculosis has proven to be a suitable model system to study the intersection of innate and adaptive immunity mechanisms with autophagy, opening many questions: How do autophagosomes/autolysosomes kill mycobacteria? How do Th1 and Th2 cytokines, such as IFN-γ or IL4/ IL-13, induce or downregulate autophagy? What is the mechanism of action of murine IRG proteins, e.g., Irgm1 involved in autophagy? Is a similar activity present in human cells? How is the spacious autophagosome formed? These can be added to the long-standing questions in the field of autophagy, such as where the membranes for autophagosomes, in particular the large ones formed during immunophagy, come from? Do bacteria have defense systems against autophagy, e.g., escape on actin tails from being trapped by isolation membranes, or inhibition of PI3P? The answers to these and other related questions will improve our understanding of immunophagy as a newly recognized host defense process and will contribute to our knowledge of the fundamental aspects of autophagy in mammalian cells. References 1. Vergne I, Chua J, Singh SB, Deretic V. Cell biology of mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 2004; 20:367-94. 2. Russell DG. Mycobacterium tuberculosis: Here today, and here tomorrow. Nat Rev Mol Cell Biol 2001; 2:569-77. 3. Ramachandra L, Noss E, Boom WH, Harding CV. Processing of Mycobacterium tuberculosis antigen 85B involves intraphagosomal formation of peptide-major histocompatibility complex II complexes and is inhibited by live bacilli that decrease phagosome maturation. J Exp Med 2001; 194:1421-32. 4. Via LE, Deretic D, Ulmer RJ, Hibler NS, Huber LA, Deretic V. Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J Biol Chem 1997; 272:13326-31. 5. Fratti RA, Backer JM, Gruenberg J, Corvera S, Deretic V. Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest. J Cell Biol 2001; 154:631-44.

www.landesbioscience.com

6. Vieira OV, Harrison RE, Scott CC, Stenmark H, Alexander D, Liu J, Gruenberg J, Schreiber AD, Grinstein S. Acquisition of Hrs, an essential component of phagosomal maturation, is impaired by mycobacteria. Mol Cell Biol 2004; 24:4593-604. 7. Simonsen A, Lippe R, Christoforidis S, Gaullier JM, Brech A, Callaghan J, Toh BH, Murphy C, Zerial M, Stenmark H. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 1998; 394:494-8. 8. Kauppi M, Simonsen A, Bremnes B, Vieira A, Callaghan J, Stenmark H, Olkkonen VM. The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane trafficking. J Cell Sci 2002; 115:899-911. 9. Vieira OV, Botelho RJ, Rameh L, Brachmann SM, Matsuo T, Davidson HW, Schreiber A, Backer JM, Cantley LC, Grinstein S. Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J Cell Biol 2001; 155:19-25. 10. Stack JH, Emr SD. Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities. J Biol Chem 1994; 269:31552-62. 11. Fratti RA, Chua J, Vergne I, Deretic V. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proc Natl Acad Sci USA 2003; 100:5437-42. 12. Chua J, Deretic V. Mycobacterium tuberculosis reprograms waves of phosphatidylinositol 3-phosphate on phagosomal organelles. J Biol Chem 2004; 279:36982-92. 13. Vergne I, Chua J, Lee HH, Lucas M, Belisle J, Deretic V. Mechanism of phagolysosome biogenesis block by viable Mycobacterium tuberculosis. Proc Natl Acad Sci USA 2005; 102:4033-38. 14. Purdy GE, Owens RM, Bennett L, Russell DG, Butcher BA. Kinetics of phosphatidylinositol-3-phosphate acquisition differ between IgG bead-containing phagosomes and Mycobacterium tuberculosis-containing phagosomes. Cell Microbiol 2005; 7:1627-34. 15. Vergne I, Chua J, Deretic V. Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade. J Exp Med 2003; 198:653-9. 16. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119:753-66. 17. Dengjel J, Schoor O, Fischer R, Reich M, Kraus M, Muller M, Kreymborg K, Altenberend F, Brandenburg J, Kalbacher H, Brock R, Driessen HG. Autophagy promotes MHC class II presentation of peptides from intracellular source proteins. Proc Natl Acad Sci USA 2005; 102:7922-7. 18. Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, Munz C. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 2005; 307:593-6. 19. Scarlatti F, Bauvy C, Ventruti A, Sala G, Cluzeaud F, Vandewalle A, Ghidoni R, Codogno P. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and upregulation of beclin 1. J Biol Chem 2004; 279:18384-91. 20. Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005; 122:927-39. 21. Lavieu G, Scarlatti F, Sala G, Carpentier S, Levade T, Ghidoni R, Botti J, Codogno P. Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation. J Biol Chem 2006; 281:8518-27. 22. Hoyer-Hansen M, Bastholm L, Mathiasen IS, Elling F, Jaattela M. Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death. Cell Death Differ 2005; 12:1297-309. 23. Garg SK, Volpe E, Palmieri G, Mattei M, Galati D, Martino A, Piccioni MS, Valente E, Bonanno E, De Vito P, Baldini PM, Spagnoli LG, Colizzi V, Fraziano M. Sphingosine 1-phosphate induces antimicrobial activity both in vitro and in vivo. J Infect Dis 2004; 189:2129-38. 24. Anes E, Kuhnel MP, Bos E, Moniz-Pereira J, Habermann A, Griffiths G. Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria. Nat Cell Biol 2003; 5:793-802. 25. Hmama Z, Sendide K, Talal A, Garcia R, Dobos K, Reiner NE. Quantitative analysis of phagolysosome fusion in intact cells: Inhibition by mycobacterial lipoarabinomannan and rescue by an 1alpha,25-dihydroxyvitamin D3-phosphoinositide 3-kinase pathway. J Cell Sci 2004; 117:2131-40. 26. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zugel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science 2006; 311:1770-3. 27. de Chastellier C, Thilo L. Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles. Cell Microbiol 2006; 8:242-56. 28. Manca C, Reed MB, Freeman S, Mathema B, Kreiswirth B, Barry IIIrd CE, Kaplan G. Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis. Infect Immun 2004; 72:5511-14. 29. Arico S, Petiot A, Bauvy C, Dubbelhuis PF, Meijer AJ, Codogno P, Ogier-Denis E. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 2001; 276:35243-6. 30. Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J Cell Biol 2002; 157:455-68. 31. Deretic V. Autophagy in innate and adaptive immunity. Trends Immunol 2005; 26:523-8.

Autophagy

177


32. Via LE, Fratti RA, McFalone M, Pagan-Ramos E, Deretic D, Deretic V. Effects of cytokines on mycobacterial phagosome maturation. J Cell Sci 1998; 111:897-905. 33. Bekpen C, Hunn JP, Rohde C, Parvanova I, Guethlein L, Dunn DM, Glowalla E, Leptin M, Howard JC. The interferon-inducible p47 (IRG) GTPases in vertebrates: Loss of the cell autonomous resistance mechanism in the human lineage. Genome Biol 2005; 6:R92. 34. MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFNgamma-inducible LRG-47. Science 2003; 302:654-9. 35. Ghosh A, Uthaiah R, Howard J, Herrmann C, Wolf E. Crystal structure of IIGP1: A paradigm for interferon-inducible p47 resistance GTPases. Mol Cell 2004; 15:727-39. 36. Klionsky DJ. The molecular machinery of autophagy: Unanswered questions. J Cell Sci 2005; 118:7-18. 37. Taylor GA, Stauber R, Rulong S, Hudson E, Pei V, Pavlakis GN, Resau JH, Vande Woude GF. The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding. J Biol Chem 1997; 272:10639-45. 38. Martens S, Sabel K, Lange R, Uthaiah R, Wolf E, Howard JC. Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: Retargeting to plasma membrane induced by phagocytosis. J Immunol 2004; 173:2594-606. 39. Kaiser F, Kaufmann SH, Zerrahn J. IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3. J Cell Sci 2004; 117:1747-56. 40. Walenta JH, Didier AJ, Liu X, Kramer H. The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins. J Cell Biol 2001; 152:923-34.

178

Autophagy

2006; Vol. 2 Issue 3