Die in vivo Bedeutung lysosomaler Membranproteine bei der intrazellulären Verwertung von Parasiten und Bakterien nach Infektion
Zusammenfassung der Projektergebnisse
Lysosomes are the primary catabolic compartments of eukaryotic cells. They degrade extracellular material that has been internalized by endocytosis and intracellular components that have been sequestered by autophagy. In addition, specialized cells contain lysosome-related organelles that store and secrete proteins for cell-type-specific functions. The functioning of a healthy cell is dependent on the proper targeting of newly synthesized lysosomal proteins. Accumulating evidence suggests that there are multiple lysosomal delivery pathways that together allow the regulated and sequential deposition of lysosomal components. The importance of lysosomal trafficking pathways is emphasized by recent findings that reveal new roles for lysosomal membrane proteins in cellular physiology and in an increasing number of diseases that are characterized by defects in lysosome biogenesis. Using a number of knockout mouse mutants we were able to underline the important role of lysosomal hydrolases but also of abundant lysosomal membrane proteins for autophagy, phagocytosis, cholesterol homeostasis and lysosomal biogenesis. Lysosomes are active organelles in the majority of professional phagocytes and in non-phagocytic cells. Intracellular defense against invading pathogens requires in most instances a functional lysosomal compartment. This is exemplified by an enhanced susceptibility to Listeria monocytogenes infection of fibroblasts and bone-marrow macrophages and increased intraphagosomal viability of bacteria in fibroblasts isolated from cathepsin D-deficient mice compared with wild type mice. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers. The crucial role of the aspartyl-protease cathepsin-D for nonoxidative listericidal defense was further supported by prolonged survival of L. monocytogenes in Ctsd-deficient mice and cells after infection. Based on infection experiments with mutant bacteria, in vitro degradation, and immunoprecipitation experiments a role of cathepsin D for the proteolytic degradation of the main bacterial virulence factor listeriolysin O was suggested. The above mentioned association between autophagy and phagocytosis is also underlined by our recent studies where cells lacking either one or both LAMPs show a disturbed autophagosome maturation. Phagolysosome biogenesis was also analyzed in these cells. Despite the fact that LAMP-1 and LAMP-2 are possibly the most abundant integral membrane proteins of phago-lysosomes, their role in phagosome function has remained elusive. Our observations suggest that LAMPs are essential for successful completion of phagosome maturation, specifically, for the transition from early to late phagosomes. In addition, we noted that the absence of both LAMP-1 and LAMP-2 delayed transport of fluid-phase markers from early endosomes to lysosomes. These observations, together with the reported requirement of LAMPs for proper formation of autolysosomes, highlight an important role for LAMPs in the dynamics of the endocytic pathway. Whereas macrophages and fibroblasts from single-LAMP-deficient mice display normal fusion of lysosomes with phagosomes, in LAMP double knockout fibroblasts phagosomes are unable to recruit late endosomal/lysosomal markers and phagocytosis is arrested prior to the acquisition of Rab7. Interestingly, the maturation of Neisseria-containing phagosomes is also disturbed and cells lacking both LAMP proteins fail to kill the engulfed pathogen. The maturation block caused by LAMP deficiency is at least partially due to the inability of (auto)phagosomes to undergo dynein/dynactin-mediated centripetal displacement along microtubules and therefore an inability to move towards lysosomes. Interestingly in LAMP-2 knockout mice an impaired phagosomal maturation in neutrophils was observed. The impairment of this important innate immune defense process in these mice leads to periodontitis. Inflammatory periodontal diseases constitute one of the most common infections in man, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils (PMNs) are an essential component of the innate immune system. We observed that LAMP-2 knockout mice developed severe periodontitis early in life. This was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes/lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient anti-microbial activity in PMNs which is needed to prevent periodontal disease. Neutrophils of the LAMP-2 knockout mice were also described earlier to contain an accumulation of autophagic vacuoles. Taken together our observations strongly suggest that an intact fusion machinery is required to successfully complete both autophagosome but also phagosome maturation. Similar to the above mentioned role of cathepsin-D for cellular defense against Listeria infections we observed an unexpected role of cathepsin-K, a lysosomal protease formerly shown to be of major importance in bone resorption due to its high expression in osteoclasts, in innate immune response to pathogen DNA. Inhibition or deficiency of cathepsin K could suppress autoimmune inflammation in experimental arthritis and experimental encephalomyelitis suggesting a general role of this protease in the regulation of inflammation. Cathepsin-K inhibition almost eliminated the signal function of toll-like receptor 9 in dendritic cells. In an independent collaborative study we could show that lysosomal proteins are of general interest for the proper function of toll like receptors in dendritic cells. Toll like receptor 4 has been reported to inhibit the lysosome-dependent degradation of phagosomes. To confirm the involvement of lysosomal degradation in the TLR4 dependent processing of dying tumor cell antigens, Lamp2-deficient dendritic cells were analyzed. Inhibition of TLR4 by TLR4Fc failed to inhibit the presentation of antigen from dying cells by Lamp2 knockout dendritic cells in conditions in which TLR4Fc did inhibit antigen presentation by wildtype-dendritic cells. These data confirm that TLR4 regulates the processing/presentation of tumor cell antigens by dendritic cells, presumably by inhibiting the lysosomal destruction of antigen.
Projektbezogene Publikationen (Auswahl)
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(2006) Cutting edge: a novel nonoxidative phagosomal mechanism exerted by cathepsin-D controls Listeria monocytogenes intracellular growth. J Immunol. 176:1321-1325
del Cerro-Vadillo E, Madrazo-Toca F, Carrasco-Marín E, Fernandez-Prieto L, Beck C, Leyva-Cobián F, Saftig P, Alvarez-Dominguez C
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(2007) Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2. Cell Microbiol. 9:2153-2166
Binker MG, Cosen-Binker LI, Terebiznik MR, Mallo GV, McCaw SE, Eskelinen EL, Willenborg M, Brumell JH, Saftig P, Grinstein S, Gray-Owen SD
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(2007) LAMP proteins are required for fusion of lysosomes with phagosomes. EMBO J. 26:313-324
Huynh KK, Eskelinen EL, Scott CC, Malevanets A, Saftig P, Grinstein S
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(2007) Tolllike receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat. Med. 13:1050-1059
Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, Mignot G, Maiuri MC, Ullrich E, Saulnier P, Yang H, Amigorena S, Ryffel B, Barrat FJ, Saftig P, Levi F, Lidereau R, Nogues C, Mira JP, Chompret A, Joulin V, Clavel-Chapelon F, Bourhis J, André F, Delaloge S, Tursz T, Kroemer G, Zitvogel L
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(2008) Autophagy: A lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta, 1793, 664-673
Eskelinen EL, Saftig P
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(2008) Cathepsin K- dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319:624-627
Asagiri M, Hirai T, Kunigami T, Kamano S, Gober HJ, Okamoto K, Nishikawa K, Latz E, Golenbock DT, Aoki K, Ohya K, Imai Y, Morishita Y, Miyazono K, Kato S, Saftig P, Takayanagi H
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(2008) Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice. J. Immunol. 180:475-482
Beertsen W, Willenborg M, Everts V, Zirogianni A, Podschun R, Schröder B, Eskelinen EL, Saftig P
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(2008) LAMP-2: a control step for phagosome and autophagosome maturation. Autophagy 4:510-2
Saftig P, Beertsen W, Eskelinen EL
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(2009) Lysosomal biogenesis and lysosomal membrane proteins: trafficking meets function. Nat. Rev. Mol. Cell Biol., 10, 623-635
Saftig, P. & Klumperman, J.
