Cholesterol depletion resulted in fusion of mycobacterium-containing phagosomes with lysosomes
Phagocytic uptake of mycobacteria depends on a normal abundance of cholesterol in the plasma membrane. Upon cholesterol depletion, mycobacteria, in contrast to other bacilli, are no longer phagocytosed by macrophages (Gatfield and Pieters, 2000; Peyron et al., 2000). Our present results now indicate that cholesterol is also required for mycobacteria to prevent fusion of phagosomes with lysosomes. Cholesterol is essential for the formation of lipid rafts, which are special membrane domains where lipid-anchored membrane proteins and sphingolipids are enriched (reviewed in Brown and London, 1998; Lai, 2003). Cholesterol-rich membrane domains harbour signalling molecules (reviewed in Manes et al., 2003) and docking molecules for membrane fusion (SNAREs; Chamberlain and Gould, 2002). A lack of cholesterol blocks in vitro homotypic fusion of yeast vacuoles (Kato and Wickner, 2001). The recruitment of lipid rafts to the phagosome membrane is involved in phagosome maturation and can be interfered with by intra-cellular pathogens (Dermine et al., 2001).
The most likely explanation for how cholesterol depletion incapacitates the ability of mycobacteria to inhibit phagosome maturation, will involve its effect on sphingolipids and related kinases. These molecules could represent a target for pathogenic mycobacteria to interfere with phagosome maturation (reviewed in Koul et al., 2004). Several molecular mechanisms for how mycobacteria prevent phagosome maturation, and therewith fusion with lysosomes, have been proposed (reviewed in, e.g. Russell, 2001; Chua et al., 2004). Events of phagosome maturation which are affected by mycobacterial interference are summarized schematically in Fig. 11 and serve to point out possible sites where cholesterol may be involved. Phagocytosis of mycobacterium blocks sphingosine kinase (SK; cf. Fig. 11 point 1). Intact lipid rafts, and therefore sufficient cholesterol, may be required for this block. Rab5 on mycobacterium-containing phagosomes (Via et al., 1997; Clemens et al., 2000) can promote phagosome maturation by a phosphatidyl inositol-3 kinase (PI3K)-independent process (Vieira et al., 2003), possibly circumventing the block exerted by the mycobacterial lipid ManLAM (cf. Fig. 11, point 3). However, mycobacterium introduces a second block by stimulating a protein kinase (p38 MAPK) which keeps Rab5 in its GDP-bound form (Fratti et al., 2003; cf. Fig. 11, point 7). Assuming this block involves signalling events that depend on intact lipid rafts, it might be overcome by cholesterol depletion and result in maturation of mycobacterium-containing phagosomes.
Figure 11. Schematic representation to summarize events during phagosome maturation which are affected by mycobacteria. 1. Receptor-mediated phagocytic uptake is accompanied by an increase in the cytosolic Ca++-concentration. Sphingosine kinase (SK) phosphorylates the membrane lipid sphingosine, which is a ligand for a G-protein coupled receptor (GPCR) involved in mediating an increase in the cytosolic calcium concentration. Phagocytosis of mycobacterium leads to a block of SK (Malik et al., 2003; Kusner, 2005). SK also appears to associate directly with the phagosome and this process is inhibited by mycobacteria (Thompson et al., 2005). 2. The released Ca++ binds to calmodulin, which then associates with a class III phosphatidyl inositol-3 kinase (PI3K), an essential enzyme for phagosome maturation (Vieira et al., 2001; reviewed in Botelho et al., 2004). 3. Ca++-associated PI3K is essential for phagosome maturation and binds to the phagosome membrane via Rab5. The latter is retained on mycobacterium-containing phagosomes (Via et al., 1997; Clemens et al., 2000). PI3K catalyses the conversion of phosphatidyl inositol (PI) to phosphatidyl inositol 3-phosphate (PI3P). The similarity between PI3P and the mycobacterial cell-wall lipid, ManLAM, is thought to interfere with this process (Fratti et al., 2001). 4. Because PI3P is required to stabilize the association of early endosome antigen 1 (EEA1) with the phagosome membrane (Simonsen et al., 1998), EEA1 is not recruited to the mycobacterium-containing phagosome membrane. 5. EEA1 acts as a tethering molecule which is involved in SNARE-mediated organellar fusion. 6. Initially, EEA1 becomes associated with the phagosome membrane only via Rab5. 7. A PI3K-independent process, by which Rab5 can lead to phagosome maturation (Vieira et al., 2003), is also blocked by mycobacteria in that they stimulate a protein kinase (p38 MAPK) which leads to the inactivation of Rab5, keeping it in its GDP-bound form (Fratti et al., 2003).
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When mycobacteria are phagocytosed in clumps and the resultant phagosome contains more than one mycobacterium, these ‘social’ phagosomes invariably mature and fuse with lysosomes, whereas ‘loner’ phagosomes in the same cells are prevented from maturing (de Chastellier et al., 1995; Clemens and Horwitz, 1995; de Chastellier and Thilo, 1997; Pietersen et al., 2004). Therefore, the inhibition of phagosome maturation does not involve cell signalling in general, but is phagosome-autonomous. This local restriction is difficult to reconcile with the proposed role of mycobacterial lipids in the prevention of phagosome maturation (Fig. 11, point 3) as such lipids have been observed to diffuse to other endocytic membranes and even to uninfected cells in proximity of infected ones (Beatty et al., 2000). Evidence that cholesterol depletion interfered with a biological process, rather than simply changing the physical properties of the phagosome membrane, was obtained by our observation that the effects of cholesterol depletion remained restricted to mycobacterium-containing phagosomes and did not affect phagosomes with inert hydrophobic latex beads when these were also present in mycobacterium-infected macrophages. Such beads were chosen here as control phagocytic particles because, in contrast to beads with a hydrophilic surface, they also block phagosome maturation, although temporarily (up to at least 3 h, de Chastellier and Thilo, 1997; see also solid squares in Fig. 4A and below). Within 12–24 h, such beads end up in phagolysosomes, after repeated events of fusion and fission with lysosomes, referred to as the ‘kiss-and-run’ mechanism (Desjardins, 1995). We observed evidence that cholesterol depletion might interfere with the fission aspect of this process: the bead-containing phagosomes remained connected to lysosomes through thin tube-like connections (Fig. 5D and E).
Previously, we have proposed that a necessary requirement for mycobacterium's ability to prevent phagosome maturation is an all-around close apposition of the phagosome membrane to the bacterial surface (de Chastellier et al., 1995; de Chastellier and Thilo, 1997). This proposal can serve to explain the maturation of social phagosomes because the phagosome membrane is distant from the bacterial surface where it spans regions between adjacent mycobacteria. In this regard, it was of special interest to note that one of the first effects of cholesterol depletion on mycobacterium-containing loner phagosomes was a partial loosening of the phagosome membrane from the mycobacterial surface, seen as an undulating limiting membrane (Fig. 2B). This was rapidly followed by fusion with lysosomes (Figs 2C, D and 3). Surprisingly, cholesterol depletion had no immediate effect on the morphology of phagosomes with hydrophobic latex beads (see Fig. 5), which started to fuse with lysosomes only about 2 h later (Fig. 4, solid circles). Cholesterol depletion might affect specific interactions of the mycobacterial surface with proteins of the phagosome membrane, conceivably by interfering with their lateral distribution in the absence of lipid rafts. How can an all-around close apposition of the phagosome membrane interfere with its maturation? We have argued that maturation requires efficient recycling from the phagosome, that this depends on the ability of the membrane to undergo tubule formation, and would therefore be severely restricted as long as the membrane remains closely apposed to the surface of the phagocytic particle (de Chastellier and Thilo, 1997). This is directly supported by comparing the rate of recycling of the transferrin receptor, which occurs with a half-life of about 1.5 min from the tubular extensions of early endosomes (Stoorvogel et al., 1989), but slowly from mycobacterium-containing phagosomes in that they lose transferrin receptor over a period of several hours (Clemens and Horwitz, 1996). Tubule formation of the phagosome is also involved in its interaction with late endocytic organelles (Harrison et al., 2003). The v-SNARE protein, cellubrevin, which is required for recycling from early endosomes, is degraded on the mycobacterium-containing phagosome (Fratti et al., 2002), a process that might require close proximity with the mycobacterial surface. The loss of cellubrevin may account for the selective local interference with efficient recycling and, therewith, phagosome maturation. In contrast, loss of cellubrevin will not prevent ongoing fusion with early endosomes as this does not depend on cellubrevin (Link et al., 1993). Cellubrevin is a v-SNARE and may be localized to cholesterol-rich microdomains known for their association with SNAREs (Lang, 2003). Cholesterol depletion could undo this localization in a way that interfered with degradation of cellubrevin by mycobacteria.
Cholesterol depletion induced phagolysosome-derived autophagy
The present study showed that when cholesterol depletion caused fusion of mycobacterium-containing phagosomes with lysosomes, the resulting phagolysosomes engaged in autophagy. These curled themselves around cytoplasmic contents, occasionally including other phagosomes, which became engulfed into an organelle of phagolysosomal origin (Fig. 6). To the best of our knowledge, this type of autophagy has not been described before and may be specific for mycobacterium-containing phagolysosomes because it did not occur for phagolysosomes with latex beads in the same infected cells. Intracellular pathogens have been linked to autophagy on numerous occasions, either when autophagy serves as a host response for bacterial destruction, or as a compartment for intra-cellular survival of the pathogens (reviewed by Dorn et al., 2002; Kirkegaard et al., 2004; Gorvel and de Chastellier, 2005; Levine, 2005). In mammalian cells, autophagy can be induced by starvation and the membrane of the autophagic vacuoles can be derived from the smooth endoplasmic reticulum (Dunn, 1990), or from other unidentified sources (Stromhaug et al., 1998; Mizushima et al., 2001). In these previous studies, the (phago-) lysosomes are not the primary source of membrane for the nascent autophagic vacuole, which only acquire lysosomal characteristics upon fusion with lysosomes to become autolysosomes (reviewed in Mizushima et al., 2002). Coxiella-containing phagolysosomes exhibit molecular markers normally associated with auto-phagosomes (reviewed in Kirkegaard et al., 2004), but autophagy itself has not been observed for such organelles. A detailed morphological study of the response of Schwann cells to cholesterol depletion as a result of exposure to tellurium has not revealed lysosome-based autophagy (Calle et al., 1999).
Autophagy by mycobacterium-containing phagolysosomes in response to cholesterol depletion could not be induced by starvation and is difficult to explain in molecular terms. Macro-autophagy in mammalian cells is controlled by diverse signalling mechanisms (reviewed by Petiot et al., 2002) and is associated with cholesterol-rich membrane domains (reviewed in Manes et al., 2003). Conceivably, cholesterol depletion could interfere with this control network. Class III PI3K is required for macro-autophagy (Petiot et al., 2002), but this enzyme may be affected by the mycobacterial lipid ManLAM (cf. Fig. 11, point 3). Therefore, either this requirement is bypassed by cholesterol depletion, or, more likely, mycobacteria-derived autophagy upon cholesterol depletion depends on other factors than those involved in macro-autophagy in general.
Upon cholesterol replenishment, mycobacterium remodels auto-phagolysosomes into immature phagosomes
It is not known to what extent delivery of mycobacterium into a phagolysosome leads to its ultimate destruction or merely inhibits its proliferation. Considering the tendency of M. tuberculosis for clumping, it is likely that most of these bacteria will be phagocytosed into a social phagosome that fuses with lysosomes (see above). For a successful infection, it must be assumed, however, that mycobacteria are not being killed when in a phagolysosome. Accordingly, we have proposed that pathogenic mycobacteria can ‘rescue’ themselves from phagolysosomes and revert into phagosomes which remain immature, which cannot fuse with lysosomes, and in which mycobacteria can multiply (de Chastellier and Thilo, 1999; Pietersen et al., 2004). The present observations now showed that when cholesterol was replenished, mycobacteria were able to rescue themselves from auto-phagolysosomes and revert to residing individually in phagosomes with the phagosome membrane in an all-around close apposition to the mycobacterial surface (Fig. 9). Furthermore, residence in the harmful environment of auto-phagolysosomes did not affect the level or the subsequent intra-phagosomal growth rate of mycobacteria (Fig. 10). We have shown previously that the phagosome membrane which surrounds mycobacteria acquires a special composition in terms of cell surface-derived glycoconjugates and proposed that this may reflect the involvement of some of these membrane proteins in establishing the close interaction with the mycobacterial surface (de Chastellier and Thilo, 2002; Pietersen et al., 2004). Considering that the bulk of internalized cell-surface glycoconjugates is recycled to the plasma membrane at a prelysosomal stage (Thilo et al., 1995) and that only a few percent become part of the lysosomal membrane (Haylett and Thilo, 1986), the present observation of a slow reestablishment of a closely apposed membrane implies that mycobacteria should actively recruit such molecules for binding to their surface, possibly in a zippering-like process.