And the Pathogenic Case

Various serious diseases, like tuberculosis or legionnaires' disease (this book gives many other examples), are caused by intracellular pathogens that are not killed by macrophages. In many cases this malfunction is thought to originate from bacteria-induced alteration of phagosome maturation, but the exact mechanism is poorly understood. Analysis of traffic alterations by pathogenic bacteria demands examination of two components: the vesicle fusion machinery of the phagocyte, which enables phagosome maturation and degradation of engulfed particles, and the pathogen interfering with this machinery.

To dissect which part of the fusion machinery is being manipulated by a pathogen, cell-free fusion assays can be used because they allow mixing of components from different cells. Using this modular principle Naroeni et al. [19] analyzed maturation of phagosomes containing Brucella suis, a pathogen that blocks fusion ofits phagosomes with lysosomes. Brucella phagosomes and lysosomes do not bind to each other, even if both lysosomes and cytosolic proteins are from non-infected cells. This suggests that it is not fusion but upstream targeting of the phagosome that is disturbed and that inhibition of phagosome-lysosome targeting is caused by modifications of the phagosome membrane. Moreover, this in vitro experiment suggested that no secreted bacterial effectors are involved when seen together with data from coinfection experiments in vivo, in which the phagosome maturation block was restricted to Brucella phago-somes and maturation of latex bead phagosomes in the same cell was undisturbed.

Experiments analyzing traffic alterations caused by Salmonella enterica or Trypanosoma cruzi revealed a very different picture. Cytosol derived from cells infected with either pathogen did not support cell-free homotypic fusion of early endosomes, suggesting that it was not the phagosomes themselves, but bacterial factors secreted into the cytosol that were responsible for inhibited vesicle fusion [25, 26]. In the case of Salmonella, the type III secretion system effector protein spiC was reported to manipulate trafficking.

There are also some hints that a soluble bacterial factor is at least in part responsible for arresting maturation of Mycobacterium-containing phagosomes at an early stage in a neutrophil model. Use of cytosol derived from cells infected with M. smegmatis or simply cytosol preincubated with these bacteria in membrane fusion experiments resulted in decreased fusion oflysosomes with phagosomes containing inert latex beads [15]. In a similar experimental setting, Vergne etal. demonstrated an inhibitory effect of the purified mycobacterial phosphatase SapM on phagosome-late endosome fusion. Based on this finding the authors hypothesize that this enzyme alters the phosphorylation status of certain lipids on the phagosomal membrane, which in turn retards recruitment of eukaryotic proteins essential for membrane fusion [27].

Cell-free systems were not only used to analyze the involvement of certain bacterial factors in trafficking alterations, but also to investigate their mechanism of action: The mycobacterial lipid PIM (phosphatidylinositol-mannoside) is thought to be essential for maintaining fusion competence of Mycobacterium-containing phagosomes with early endosomes. Using in vitro fusion systems, Vergne et al. [21] investigated the effects caused by PIM in two respects. First, they demonstrated that PIM action stimulates homotypic fusion of early but not late endosomes. Second, they narrowed down the site of action of PIM, showing that this stimulatory effect was present only under conditions in which Rab proteins were limiting for organelle fusion.

In vitro experiments according to the modular principle have been used not only to investigate the action of pathogenicity factors, but also to study the immune activation of phagocytes in response to infection: Peyron et al. [15], again in a neutrophil model, demonstrated an increased fusion rate of phagosomes and lysosomes in the presence of cytosol from activated macrophages. This is particularly interesting as recent work has suggested that overall phagosome-lysosome fusion is, if anything, slower in activated than in resting macrophages [28]. Mukherjee et al. [29] recreated in vitro the altered trafficking of Salmonella-containing phagosomes after immune acitivation, and found decreased fusion competence ofthese phagosomes with early endosomes but increased fusion competence with lysosomes. Experiments combining phago-somes and cytosol coming either from activated or resting cells revealed that either component is influenced by immune activation: Fusion of early phagosomes and early endosomes is decreased, even if only one element is derived from activated cells; this effect is enhanced when both phagosomes and cytosol are from activated cells.

Cell-free fusion assays have been a useful tool in the investigation of both the mechanism of phagosome maturation in general and traffic alterations by pathogenic bacteria. But up till now, no systematic reconstitution of fusion of pathogen-containing phagosomes with some endocytic organelles but not with others has been done. Such an approach will be very valuable to further illuminate the microbial toolbox that allows intracellular pathogens to circumvent delivery to lysosomes. Such an in vitro approach will be particularly valuable in revealing the mechanism and details of interference of bacterial effector proteins with defined factors of the eukaryotic vesicle fusion machinery.

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