II. C Internalization of bacteria and maturation of the phagosome
II. C. 1. Phagocytosis in mammalian phagocytes
During my PhD I studied the mechanisms of adhesion of the phagocyte to bacteria and the intracellular killing of bacteria in the phagolysosome. I did not investigate the specific aspects of the phagosome formation and its subsequent maturation, thus I will describe rapidly here the mechanisms at play focusing on the well described examples of the Fc!R and CR3 mediated phagocytosis in mammals and make the parallel in Dictyostelium.
a. Fc!Rs- and CR3-mediated phagocytosis
Fc!Rs and CR3 have been shown to trigger specifically phagocytosis (Aderem and Underhill, 1999).
The initial step in phagocytic cup formation differs between these two receptors, as evidenced by the different shapes of the phagocytic cups: Fc!Rs induce membrane extensions and a zipper-like engulfment, CR3s induce a sinking-like internalization of bacteria (Cougoule et al., 2004; Tollis et al., 2010). Identification of proteins involved in this initial step revealed clear differences.
Activation of Fc!Rs triggers phosphorylation events performed by Src and Syk protein kinases.
Phosphorylation occurs in the cytoplasmic tail of the receptor, which leads to the activation of two classes of proteins: members of Rho family GTPases and lipid modifying enzymes. The small GTPases Rac1 and Cdc42 as well as the class I phosphatidylinositol 3’-kinase (PI3K) and the phospholipase C (PLC) are major actors of the phagosome formation (Fig. 11). The most abundant phosphoinositides at the plasma membrane are PI(4,5)P2, they are converted at the site of phagocytosis into PI(3,4,5)P3 by class I PI3K and into diacylglycerol (DAG) and I(1,4,5)P3 by PLC (Minakami et al., 2010). Modified lipids have the ability to activate downstream signaling. For example PI(3,4,5)P3 binds to the pleckstrin homology (PH) domain of small GTPases and activate them. Lipid modifying enzymes and GTPases act synergystically and regulate the recruitment of nucleation-promoting factors (WASP/WAVE) and of actin nucleating Arp2/3 proteins complexe (Bohdanowicz et al., 2010; Groves et al., 2008) (Fig. 11). In contrast, activation of CR3 does not involves phosphorylation events but triggers direct activation of lipid modifying enzymes (PI3K, PLC) and RhoA, a member of the Rho family of GTPases (Dupuy and Caron, 2008). RhoA mediates the outside-in signaling through at least two effectors: the Rock serine threonine kinase and the mDia formin-related elongating factor. Rock activates Arp2/3 and mDia acts in concert with the Rock/Arp2/3 complex to drive actin polymerization (Dupuy and Caron, 2008).
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Figure 11: Fc!R- and CR3-mediated phagocytosis.
Fc!Rs and CR3 are phagocytic receptors, which trigger bacteria engulfment. Fc!Rs triggers zipper-like engulfment while CR3 triggers sinking-zipper-like engulfment. Fc!R triggers phosphorylation events mediated by Src and Syk kinases, leading to the activation of lipid modifying enzymes (e. g. PI3K) as well as Rho family GTPases (e. g. Rac1 and Cdc42). CR3 activation recruits directly lipid modifying enzymes and the small Rho GTPase RhoA. Both phagocytic processes require the actin nucleating proteins Arp2/3 and lead to actin polymerization and bacteria engulfment.
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In both situations, polymerization of actin into actin filaments plays a crucial role in phagosome formation. However it is not clear if actin polymerization provides the energy necessary for membrane extension or if actin polymerization acts as a stabilizer of pseudopods, while membrane extension is achieved by other mechanisms. Phagosome formation also requires new membrane input in order to allow the cell to maintain its plasma membrane surface. It has been shown that fusion of endosomes to forming phagosomes provides membrane to allow cells to engulf large particles with no reduction of their surface. The surface of phagocytes can even increase during phagocytosis (Holevinsky and Nelson, 1998).
The final step of internalization is the complete closure of the phagosome that is based on contractile activity involving dynamin and myosin proteins (Huynh et al., 2007; Swanson et al., 1999).
The newly formed phagosome matures by fusing with endosomal vesicles containing bactericidal molecules. One of the hallmarks of maturating phagosome is the depolymerization of actin, allowing the fusion of the phagosome with endocytic compartments.
b. From phagosome to phagolysosome
Between phagosome closure and bacterial degradation, a series of fusion and fission events allows the phagosome to acquire new membrane as well as lumenal molecules, necessary for bacterial killing (Desjardins, 1995; Fairn and Grinstein, 2012). The compartments of the endocytic pathway, from early endosomes to lysosomes, are major contributors to the maturing phagosome. This is illustrated by the similarities in membrane composition between early endosomes and early phagosomes, late endosomes and late phagosomes and finally between lysosomes and phagolysosomes (Fairn and Grinstein, 2012; Vieira et al., 2002) (Fig. 12). Two main types of markers of endosomal/phagosomal maturation can be distinguished.
First, the proteins involved in the fusion and fission events between endosomes and phagosomes, second, the molecules transported and transferred from endosomes to phagosomes that eventually participate in bacterial killing.
Rab proteins, members of the Ras superfamily of small GTPases, associated with their effector proteins, and the soluble, N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), are major actors during fusion and fission events between homotypic compartments (Coppolino et al., 2001; Schwartz et al., 2007; Vieira et al., 2002). For example, Rab5 and its effector protein EEA1 (Early endosome antigen 1) have been shown to mediate homotypic fusion between early endosomal compartments and newly formed phagosomes, hence Rab5/EEA1 are identified in both compartments (Vieira et al., 2001).
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Figure 12: Phagosome maturation. After engulfment of bacteria actin depolymerizes and allows homotypics fusion and fission events. These events are mediated by small Ras GTPases such as Rab5 and Rab7. Rab5 has been shown to be associated to early endosomes/phagosomes, and is exchanged with Rab7 allowing transition to late endosomes/phagosome. This scheme depicts some examples of proteins distribution along the maturation process. In addition, the maturation of endosomes/phagosomes is associated to a progressive acidification of their lumens, illustrated on the scheme by a color gradient.
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Similarly, late endosomes are characterized by the presence of Rab7 associated to its effector protein Rab7-interacting lysosomal protein (RILP) promoting fusion with late phagosome, and hence are found in the phagosomal membrane of late phagosomes (Cantalupo et al., 2001; Fairn and Grinstein, 2012). More than 20 other Rab proteins have been identified by proteomics or immunohistochemistry associated to the phagosomal membrane, their roles in phagosome maturation remain to be elucidated for the vast majority of them (Fairn and Grinstein, 2012).
Homotypic fusion and fission of endosomes with phagosomes allow the transport into phagosomes, as well as the retrieval, of molecules in a chronologically orchestrated manner. These molecules can be used as markers of phagosomal maturation. Trasnferrin receptor, a membrane receptor, which binds to extracellular transferrin, has been shown to cycle between plasma membrane and early/sorting endosomes (Vieira et al., 2002). This receptor is also found in the membrane of newly formed phagosomes but is not found in late phagosomes or in phagolysosomes. Another example is the mannose-6 phosphate receptor (M6PR), which binds to mannose-6 phosphate residues of soluble hydrolytic enzymes and is found on the membrane of late endosome/phagosome but not of lysosomes (Ghosh et al., 2003). Phagosome maturation is also characterized by the progressive acidification of the phagosomal lumen that parallels the progressive acidification of endocytic compartments. This acidification is provided by the Vacuolar H+-ATPase, which assembles at the phagosomal membrane and has been shown to be required for phagosome maturation as well as for optimal bactericidal activity in the phagosome (see Introduction II. D.). Measurements of phagosomal pH using fluorescent pH sensitive probes showed a pH above 6 in newly formed phagosome, which decreases to as low as pH 4.5 in phagolysosome (Vieira et al., 2002) (Fig. 12).
Several others proteins directly involved in bacterial killing are characteristics of phagosome maturation such as the progressive accumulation of mature hydrolases like cathepsin D as well as lysosomal associated membrane proteins (LAMPs).
II. C. 2. Bacterial uptake and phagosome maturation in Dictyostelium
Analysis of selected mutants revealed that in Dictyostelium like in mammals, internalization of particles is an actin-based mechanism (Eichinger et al., 1999). Direct visualization of the internalization of fluorescent bacteria by Dictyostelium cells expressing fluorescent-actin binding proteins revealed a highly dynamic actin cytoskeleton reorganization that involves recruitment of actin within seconds after the cell received phagocytosis stimuli (Clarke and Maddera, 2006).
Similar to mammalian phagocytes, phosphatidylinositides, lipids modifying enzymes, small GTPases and actin binding proteins are major regulators of the molecular machinery leading to the formation of the actin coat. In particular, orthologs of the mammalian PI3K, the DdPIK1 and 2,
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orthologs of the members of the Rho family of small GTPases such as Rac1, the orthologue WAVE/Scar and orthologs of the actin nucleating proteins Arp2/3 have been identified in Dictyostelium and are essential for actin-mediated particle engulfment (Eichinger et al., 1999) (Fig.
13).
After removal of the actin coat, the phagosome of Dictyostelium undergoes major modifications, caused by homotypic fusion and fission with endocytic compartments, in order to acquire bactericidal activity as described above in mammalian phagocytes (Eichinger et al., 1999). A particularity in Dictyostelium is that the phagolysosome converts into a post-lysosome that has the ability to fuse with the plasma membrane in order to expel undigested material. In mammalian cells, phagolysosomes are usually considered as dead end.
Several proteins have been identified and used as specific markers of various compartments.
Among these proteins the small GTPase Rab7 has been shown to be involved in the early step of phagosome maturation as in mammals. Newly formed phagosomes also start acquiring acid hydrolases such as cysteine proteases while acquisition of others lysosomal enzymes such as "-mannosidase occurs after 15 minutes. Kinetic analysis revealed that these enzymes are not delivered all together in one bulk, but instead are found in distinct compartments, following distinct kinetics (Cardelli et al., 1986; Souza et al., 1997).
The newly formed phagosome acidifies rapidly (1-2 min) after phagosome closure following delivery of the Vacuolar H+-ATPase. Endosomal pH can be measured by allowing cells to engulf fluorescent pH-sensitive probes such as fluoresceine or Oregon Green (Aubry et al., 1993;
Marchetti et al., 2009). Results showed that lysosome exhibits an acidic pH below 4, and that after 60 minutes, pH neutralizes to values about 6. Dictyostelium possesses a single isoform of the VatM sub-unit of the mammalian V H+-ATPase. Clarke and colleagues created a VatM-GFP fusion protein and visualized the distribution of the pump during phagocytosis of yeast particles (Clarke et al., 2002). They showed that the acquisition of VatM at the membrane of the phagosome occurred within 5-10 minutes after phagocytosis initiation and is acquired from pre-existing endosomes that fuse with maturing phagosomes. VatM is removed from the postlysosome, a mechanism that accounts for the reneutralization of the lumen of this compartment (Clarke et al., 2010). In addition to pH neutralization, postlysosomes have been shown to associate to actin-binding vacuolin B, which has been proposed to mediate fusion with the cell membrane (Bozzaro et al., 2008; Duhon and Cardelli, 2002) (Fig. 14).
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Figure 13: Internalization of bacteria in Dictyostelium is an actin based-mechanism similar to those in mammalian phagocytes. This scheme compares the major step of particle internalization by mammalian phagocyte (green line) on the left and Dictyosteliun cells on the left (red line). To date, the receptor of Dictyostelium is still unknown but adhesion of the bacteria to the cell trigger activation of lipid modifying enzyme like PLC, small GPTases like RasS, and effector of small GTPases such as Scar that promote activation of actin nucleating proteins like Arp2/3. This mechanism is similar in many aspects to the Fc!R-mediated phagocytosis depicted on the left.
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Figure 14: The phagocytic pathway in Dictyostelium. After particle is internalized, three major compartments can be detected, that correspond to three different stages of maturation of the phagosome. First the newly formed phagosome undergoes rapid modification after closure (1-2 min) such as acquisition of cysteine proteinases and V H+-ATPase. Then the phagolysosome is the more acidic compartment (pH around 5), where the burst of lysosomal enzymes is found. Finally, The lumen of the lysosome progressively neutralizes to form the post-lysosome, which acquires anchor proteins for actin filament such as vacuolin B. This compartment will fuse with the cell membrane in order to release undigested material.
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