Studying Normal Maturation

Professional phagocytic cells are central to innate immunity. They recognize and subsequently internalize microbes that enter the sterile portions of the human body. The newly formed phagosome then matures by fusion with endocytic organelles into a phagolysosome. In almost all cases this maturation process is not actively influenced by the engulfed microorganism and leads to efficient killing and degradation of the microbe. Microorganisms that are not killed by the phagocytes but rather multiply in them have in most cases evolved strategies to divert normal phagolysosome formation (see Chapter 10). To understand how these pathogens alter phagosome maturation it is essential to understand the molecular machinery involved in the undisturbed process. As a first biochemical step in this direction, fusion between endocytic compartments and phagosomes containing particles that do not interfere with normal maturation has been reconstituted. Not many apathogenic bacteria have been used in such experiments but, as decribed below, three model systems have become popular for technical reasons.

Early in vitro experiments used formaldehyde-fixed Staphylococcus aureus as phagocytic probe [9]. These bacteria bind the constant part of IgG antibodies via their surface Protein A and therefore expose free variable (Fab) parts. This feature makes them particularly suitable for the creation of an in vitro fusion assay as they can be used as affinity reagents for any molecule to which a specific antibody is available. Phagosomes containing antibody-coated S. aureus are fused with endosomes enclosing a corresponding antigen-enzyme complex (e.g., anti-dinitrophenol antibody/ dinitrophenol-derivatized b-glucuronidase). Fusion of both kinds of organelles can then be quantified as bacteria-associated enzyme activity (Figure 6.1a). An antigen-bovine serum albumin (BSA) conjugate present in the reaction buffer functions as a scavenger and impedes mutual binding of antibody-coated bacteria and antigen-enzyme complexes from broken organelles. In more recent approaches bacteria were substituted with synthetic particles of a similar size, either latex beads or paramagnetic beads [12, 21]. Their respective unique physical properties allow easy purification of bead phagosomes from cell lysates. Due to their relatively low density, latex bead phagosomes can be obtained in high purity in just one discontinuous sucrose density gradient centrifugation, while phagosomes containing magnetic particles can be isolated from cell lysates using a magnet. Furthermore, because of their uniform structure, size and shape beads can be easily identified in electron microscopy. Beads are commercially available with a variety of surface modifications, allowing covalent coupling of molecules such as avidin or biotin, making them excellent probes for in vitro fusion assays. However, inert beads do come with a price. As they are not at all degradable, their maturation features may be different from those of degradable materials. In addition, protocols established for in vitro reactions containing such handy particles are not directly useful in studies with microbial particles, as purification of the phagosome is vastly different and comes with different yields and subcellular contaminations.

Experiments in cell-free systems have great potential in the analysis of the molecular machinery involved in the fusion of phagosomes with endocytic compartments. First, such systems can be manipulated in many ways. While many experimental approaches to trafficking exist for intact cells, reaction conditions in cell-free systems or semi-intact cells can be changed readily in ways that are not possible with intact cells. For example, membrane-impermeant drugs or antibodies against relevant proteins can be added in vitro, and even ion concentrations can be adjusted. The even greater advantage of cell-free vesicle fusion systems is that they can be used to monitor an individual type of fusion step and that they can dissect the order of events, eventually leading to membrane fusion.

By purification of phagosome and endosome populations of defined ages, fusion between phagosomes and endosomes of different maturation stages can be investigated [12]. Such experiments, carried out by the group of Griffiths, revealed preference for fusion of organelles of the same maturation stage ("homotypic fusion") and indicated decreasing fusion competence with increasing organelle age. The Griffiths group also assessed the distinct involvement of actin in different phagosome maturation steps [22,23]. They showed that membranes oflate but not early phagosomes and endosomes are able to catalyze actin nucelation in vitro and that, accordingly, a correlation of actin nucleation and fusion efficiency is restricted to late endocytic and phagocytic compartments (see Chapter 8).

Membrane fusion is a multistep process, which involves directed transport of compartments along cytoskeletal filaments (targeting), a loose connection of prospective fusion partners via Rab proteins and tethering factors (tethering), followed by an extremely close connection of membranes mediated by SNARE proteins (docking) and subsequent membrane fusion. Essential to understanding the role of a molecular factor, whether it is a protein part of the eukaryotic membrane fusion machinery or a bacterial factor interfering with phagosome maturation, is identification ofthe subprocess in which it is involved. As a paradigm, yeast vacuoles can be used in the analysis of in vitro fusion studies, allowing the staging of the action of molecular factors [24]. Experimentally, this is achieved by reversible inhibition of the overall fusion reaction at a defined stage. For example, organelle docking is blocked using an anti-SNARE antibody while the fusion reaction is allowed to proceed up to the point where the block has been placed (here membrane docking). Subsequently, inhibition is reversed by addition of an excess of the corresponding antigen and concurrently an inhibitor of a functionally unknown second factor is added. If the latter drug inhibits overall fusion but does not inhibit the fusion reaction that remains after relieving SNARE inhibition, then it can be concluded that the substep of the fusion reaction inhibited by the compount had been passed while the antibody was inhibiting. Therefore, the inhibited step would be upstream of SNARE action (docking), that is, a priming or tethering reaction.

Recently, such staging experiments have been performed for phagosome-lysosome interaction in vitro. In a cell-free assay, Stockinger et al. demonstrated the differential requirements for actin, calmodulin and calcium in phagosome-lysosome targeting. Actin and calmodulin are needed to establish a complex of both organelles, then vesicle attachment becomes resistant to inhibitors of actin and calmodulin and a high calcium concentration is specifically required for stabilization of that complex [20].

A third important advantage of in vitro reconstituted membrane fusion is that most observed effects are a consequence of direct interference with the system. In living cells, knockdown of gene expression by RNA interference or gene knockout often leads to changed expression, activity and/or degradation of other factors as well. In an in vitro membrane fusion reaction, there is no transcription or translation and, therefore, effects after drug addition are much less prone to artefacts.

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