On the killing of mycobacteria by macrophages

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis , the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.     

Porins facilitate nitric oxide-mediated killing of mycobacteria

Non-pathogenic mycobacteria such us Mycobacterium smegmatis reside in macrophages within phagosomes that fuse with late endocytic/lysosomal compartments. This sequential fusion process is required for the killing of non-pathogenic mycobacteria by macrophages. Porins are proteins that allow the influx of hydrophilic molecules across the mycobacterial outer membrane. Deletion of the porins MspA, MspC and MspD significantly increased survival of M. smegmatis in J774 macrophages. However, the mechanism underlying this observation is unknown. Internalization of wild-type M. smegmatis (SMR5) and the porin triple mutant (ML16) by macrophages was identical indicating that the viability of the porin mutant in vivo was enhanced. This was not due to effects on phagosome trafficking since fusion of phagosomes containing the mutant with late endocytic compartments was unaffected. Moreover, in ML16-infected macrophages, the generation of nitric oxide (NO) was similar to the wild type-infected cells. However, ML16 was significantly more resistant to the effects of NO in vitro compared to SMR5. Our data provide evidence that porins render mycobacteria vulnerable to killing by reactive nitrogen intermediates within phagosomes probably by facilitating uptake of NO across the mycobacterial outer membrane.     

Mycobacterial manipulation of the host cell

Phagosome biogenesis, the process by which macrophages neutralize ingested pathogens and initiate antigen presentation, has entered the field of cellular mycobacteriology research largely owing to the discovery 30 years ago that phagosomes harboring mycobacteria are refractory to fusion with lysosomes. In the past decade, the use of molecular genetics and biology in different model systems to study phagosome biogenesis have made significant advances in understanding subtle mechanisms by which mycobacteria inhibit the maturation of its phagosome. Thus, we are beginning to appreciate the extent to which these pathogens are able to interfere with innate immune responses and manipulate defense mechanisms to enhance their survival within the human host cell. Here, we summarize current knowledge about phagosome maturation arrest in infected macrophages and the subsequent attenuation of the macrophage-initiated adaptive anti-mycobacterial immune defenses.     

Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles

Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.     

A coat protein on phagosomes involved in the intracellular survival of mycobacteria.

Mycobacteria are intracellular pathogens that can survive within macrophage phagosomes, thereby evading host defense strategies by largely unknown mechanisms. We have identified a WD repeat host protein that was recruited to and actively retained on phagosomes by living, but not dead, mycobacteria. This protein, termed TACO, represents a component of the phagosome coat that is normally released prior to phagosome fusion with or maturation into lysosomes. In macrophages lacking TACO, mycobacteria were readily transported to lysosomes followed by their degradation. Expression of TACO in nonmacrophages prevented lysosomal delivery of mycobacteria and prolonged their intracellular survival. Active retention of TACO on phagosomes by living mycobacteria thus represents a mechanism preventing cargo delivery to lysosomes, allowing mycobacteria to survive within macrophages.     

Mycobacterium's arrest of phagosome maturation in macrophages requires Rab5 activity and accessibility to iron

Many mycobacteria are intramacrophage pathogens that reside within nonacidified phagosomes that fuse with early endosomes but do not mature to phagolysosomes. The mechanism by which mycobacteria block this maturation process remains elusive. To gain insight into whether fusion with early endosomes is required for mycobacteria-mediated inhibition of phagosome maturation, we investigated how perturbing the GTPase cycles of Rab5 and Rab7, GTPases that regulate early and late endosome fusion, respectively, would affect phagosome maturation. Retroviral transduction of the constitutively activated forms of both GTPases into primary murine macrophages had no effect on Mycobacterium avium retention in an early endosomal compartment. Interestingly, expression of dominant negative Rab5, Rab5(S34N), but not dominant negative Rab7, resulted in a significant increase in colocalization of M. avium with markers of late endosomes/lysosomes and increased mycobacterial killing. This colocalization was specific to mycobacteria since Rab5(S34N) expressing cells showed diminished trafficking of endocytic tracers to lysosomes. We further demonstrated that maturation of M. avium phagosomes was halted in Rab5(S34N) expressing macrophages supplemented with exogenous iron. These findings suggest that fusion with early endosomes is required for mycobacterial retention in early phagosomal compartments and that an inadequate supply of iron is one factor in mycobacteria's inability to prevent the normal maturation process in Rab5(S34N)-expressing macrophages.     

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.     

Modulation of cellular phosphatidylinositol 3-phosphate levels in primary macrophages affects heat-killed but not viable Mycobacterium avium's transport through the phagosome maturation process

Most disease causing mycobacteria are intramacrophage pathogens which replicate within nonacidified phagosomes that can interact with the early endosomal network but fail to mature to a phagolysosome. The mycobacterial phagosome retain some proteins required for fusion with endocytic vesicles including Rab5 but lack others such as early endosomal autoantigen 1 (EEA1). As the membrane lipid phosphatidylinositol 3-phosphate (PtdIns-3-P) is required for EEA1 membrane association and phagosome maturation, it may be a potential target of pathogenic mycobacteria. To test this hypothesis, macrophage cellular levels of PtdIns-3-P were altered by retroviral introduction of the type III Phosphoinositide 3-Kinase (VPS34) and the PtdIns-3-P phosphatase myotubularin 1 (MTM1). By utilizing the PtdIns-3-P-specific probes FYVE and PX coupled to EGFP (EGFP-2-FYVE and EGFP-PX, respectively), the expression of PtdIns-3-P on the mycobacterial phagosome was addressed. All phagosomes containing viable Mycobacterium avium stained positive for EGFP-2-FYVE and EGFP-PX despite obvious differences in PtdIns-3-P concentrations in cells expressing MTM1 or VPS34. Altering PtdIns-3-P cellular concentrations did not affect trafficking of live bacilli. However, a significant increase in the transport of killed bacilli to a late endosomal/lysosomal compartment was observed in VPS34-compared to MTM1-transduced macrophages. Therefore, although overexpression of PdtIns-3-P in macrophages can facilitate phagosome maturation, its effect on phagosomes containing viable M. avium was negligible.     

Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria

Pathogenic mycobacteria such as Mycobacterium tuberculosis and Mycobacterium avium facilitate disease by surviving intracellularly within a potentially hostile environment: the macrophage phagosome. They inhibit phagosome maturation processes, including fusion with lysosomes, acidification and, as shown here, membrane actin assembly. An in vitro assay developed for latex bead phagosomes (LBPs) provided insights into membrane signalling events that regulate phagosome actin assembly, a process linked to membrane fusion. Different lipids were found to stimulate or inhibit actin assembly by LBPs and mycobacterial phagosomes in vitro. In addition, selected lipids activated actin assembly and phagosome maturation in infected macrophages, resulting in a significant killing of M. tuberculosis and M. avium. In contrast, the polyunsaturated sigma-3 lipids behaved differently and stimulated pathogen growth. Thus, lipids can be involved in both stimulatory and inhibitory signalling networks in the phagosomal membrane.     

Membrane trafficking along the phagocytic pathway

Phagosome maturation involves extensive remodelling of the phagosomal membrane as a result of intracellular transport events. Newly formed phagosomes exchange membrane-associated and soluble proteins with early endosomes by fusion. Budding of vesicles from the phagosome and fusion with Golgi-derived vesicles may also contribute to the remodelling of the phagosomal compartment. As a consequence of changes in membrane composition, phagosomes acquire the ability to fuse with late endocytic compartments. In vitro reconstitution and other studies suggest that the trafficking events underlying phagosome maturation require several GTP-binding proteins, including Rab5 and Galphas', NSF-SNAP-SNARE complexes and coatomers.     

Direct delivery of procathepsin D to phagosomes: implications for phagosome biogenesis and parasitism by Mycobacterium

Phagosome maturation is characterized by the sequential acquisition and loss of proteins by the phagocytic vacuole during the formation of an acidic and hydrolytic compartment where degradation of the phagocytosed particle occurs. Transfer of proteins to the maturing phagosome occurs by fusion with a range of vesicles. Here we describe direct fusion of early phagosomes with vesicles that appear to be derived from the biosynthetic pathway. In mouse bone marrow macrophages, the 51 kDa proform of cathepsin D was found in vesicles of the ER/Golgi network that could be discriminated from endosomal vesicles which in turn contained the 46 and 30 kDa processed forms of the enzyme. Procathepsin D was acquired by phagosomes formed around inert particles such as IgG-coated beads and could be "protected" by blocking acidification with Bafilomycin A1. Mycobacterium avium-containing vacuoles from established infections possessed both pro- and processed cathepsin D similar to early bead-containing phagosomes. In contrast phagosomes harboring dead mycobacteria demonstrated markedly enhanced acquisition of the 46kDa form within 4 h post internalization and only low levels of procathepsin D.     

Association of a macrophage galactoside-binding protein with Mycobacterium-containing phagosomes

Mycobacteria reside intracellularly in a vacuole that allows it to circumvent the antimicrobial environment of the host macrophage. Although the mycobacterial phagosome exhibits selective fusion with vesicles of the endosomal system, identification of host and bacterial factors associated with phagosome bio-genesis is limited. To identify these potential factors, mAbs were generated to a membrane preparation of mycobacterial phagosomes isolated from M. tuberculosis -infected macrophages. A mAb recognizing a 32–35 kDa macrophage protein associated with the phagosomal membrane of Mycobacterium was identified. N-terminal sequence analysis identified this protein as Mac-2 or galectin-3, a galactoside-binding protein of macrophages. Galectin-3 (gal-3) was shown to accumulate in Mycobacterium -containing phagosomes during the course of infection. This accumu-lation was specific for phagosomes containing live mycobacteria and occurred primarily at the cytosolic face of the phagosome membrane. In addition, bind-ing of gal-3 to mycobacterial phosphatidylinositol mannosides (PIMs) demonstrated a novel interaction between host carbohydrate-binding proteins and released mycobacterial glycolipids. Infection of macrophages from gal-3-deficient mice indicated that the protein did not play a role in infection in vitro . In contrast, infection of gal-3-deficient mice revealed a reduced capacity to clear late but not early infection.     

PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome. Phagolysosomes are highly acidic and degradative leading to particle decomposition. Phagosome maturation is intimately dependent on the endosomal pathway, during which diverse cargoes are sorted for recycling to the plasma membrane or for degradation in lysosomes. Not surprisingly, various regulators of the endosomal pathway are also required for phagosome maturation, including phosphatidylinositol‐3‐phosphate, an early endosomal regulator. However, phosphatidylinositol‐3‐phosphate can be modified by the lipid kinase PIKfyve into phosphatidylinositol‐3,5‐bisphosphate, which controls late endosome/lysosome functions. The role of phosphatidylinositol‐3,5‐bisphosphate in macrophages and phagosome maturation remains basically unexplored. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that inhibition of PIKfyve hindered certain steps of phagosome maturation. In particular, PIKfyve antagonists delayed removal of phosphatidylinositol‐3‐phosphate and reduced acquisition of LAMP1 and cathepsin D, both common lysosomal proteins. Consistent with this, the degradative capacity of phagosomes was reduced but phagosomes appeared to still acidify. We also showed that trafficking to lysosomes and their degradative capacity was reduced by PIKfyve inhibition. Overall, we provide evidence that PIKfyve, likely through phosphatidylinositol‐3,5‐bisphosphate synthesis, plays a significant role in endolysosomal and phagosome maturation in macrophages.      

Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis

Mycobacterium tuberculosis is an intracellular pathogen of macrophages and escapes the macrophages' bactericidal effectors by interfering with phagosome-lysosome fusion. IFN-γ activation renders the macrophages capable of killing intracellular mycobacteria by overcoming the phagosome maturation block, nutrient deprivation and exposure to microbicidal effectors including nitric oxide (NO). While the importance about NO for the control of mycobacterial infection in murine macrophages is well documented, the underlying mechanism has not been revealed yet. In this study we show that IFN-γ induced apoptosis in mycobacteria-infected macrophages, which was strictly dependent on NO. Subsequently, NO-mediated apoptosis resulted in the killing of intracellular mycobacteria independent of autophagy. In fact, killing of mycobacteria was susceptible to the autophagy inhibitor 3-methyladenine (3-MA). However, 3-MA also suppressed NO production, which is an important off-target effect to be considered in autophagy studies using 3-MA. Inhibition of caspase 3/7 activation, as well as NO production, abolished apoptosis and elimination of mycobacteria by IFN-γ activated macrophages. In line with the finding that drug-induced apoptosis kills intracellular mycobacteria in the absence of NO, we identified NO-mediated apoptosis as a new defense mechanism of activated macrophages against M. tuberculosis.     

Ezrin promotes actin assembly at the phagosome membrane and regulates phago-lysosomal fusion

Phagosome maturation is defined as the process by which phagosomes fuse sequentially with endosomes and lysosomes to acquire an acidic pH and hydrolases that degrade ingested particles. While the essential role of actin cytoskeleton remodeling during particle internalization is well established, its role during the later stages of phagosome maturation remains largely unknown. We have previously shown that purified mature phagosomes assemble F-actin at their membrane, and that the ezrin-radixin-moesin (ERM) proteins ezrin and moesin participate in this process. Moreover, we provided evidence that actin assembly on purified phagosomes stimulates their fusion with late endocytic compartments in vitro. In this study, we further investigated the role of ezrin in phagosome maturation. We engineered a structurally open form of ezrin and demonstrated that ezrin binds directly to the actin assembly promoting factor N-WASP (Neural Wiskott-Aldrich Syndrome Protein) by its FERM domain. Using a cell-free system, we found that ezrin stimulates F-actin assembly on purified phagosomes by recruiting the N-WASP-Arp2/3 machinery. Accordingly, we showed that the down-regulation of ezrin activity in macrophages by a dominant-negative approach caused reduced F-actin accumulation on maturing phagosomes. Furthermore, using fluorescence and electron microscopy, we found that ezrin is required for the efficient fusion between phagosomes and lysosomes. Live-cell imaging analysis supported the notion that ezrin is necessary for the fusogenic process itself, promoting the transfer of the lysosome content into the phagosomal lumen.     

Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes

Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 μm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome–lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.     

Macrophage activation downregulates the degradative capacity of the phagosome

The phagosome is key to most macrophage functions. It is the site of degradation of particulate material, of bacterial killing and the generation of peptides for antigen presentation. Despite its role at the fulcrum of the innate and acquired immune systems, little is known about the physiology of this organelle in activated macrophages. In this study, we utilize fluorometric techniques to characterize functional alterations in the lumenal environment of the maturing phagosome following stimulation of macrophages with interferon-gamma and/or lipopolysaccharide. In addition to modulating the kinetics of phagosomal acidification, activation results in a phagosome with diminished hydrolytic activities that varies markedly with the activation status of the cell. Differential levels of proteolytic, lipolytic and beta-galactosidase activities were observed in the phagosome but not in the total lysosomal extract, indicating selective delivery of enzymes to the developing phagosome. Despite the suppression of hydrolytic activities observed in early phagosomes, late phagosomes exhibit an enhanced and protracted accumulation of lysosomal cargo. The data are consistent with limiting proteolysis in the early phagosome to maximize epitope generation and antigen presentation while sequestering the degradative capacity in the late phagolysosome.     

Cytokine effects on maturation of the phagosomes containing Mycobacteria avium subspecies paratuberculosis in J774 cells

Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages. Intracellular survival of several species of pathogenic mycobacteria is dependent on inhibition of maturation of the phagosomes containing these pathogens into functional phagolysosomes. In activated macrophages, however, this capacity is reduced, leading to increased bacterial killing. It is the hypothesis of this study that there is increased acidification and maturation of the phagosome containing M. a. ptb in interferon gamma and lipopolysaccharide (IFN-gamma/LPS) activated macrophages. In activated macrophages colocalization of M. a. ptb with either a marker of acidic compartments (Lysotracker Red) or compartments containing a late phagosome maturation marker lysosome-associated membrane protein-1 (Lamp-1) were evaluated by laser confocal microscopy. Intracellular survival of M. a. ptb in activated macrophages was evaluated directly using differential fluorescent live/dead staining. The results of this study demonstrated increased colocalization of both Lysotracker Red and Lamp-1 with FITC labeled M. a. ptb, which correlated with decreased survival of M. a. ptb within activated macrophages.     

ATP-mediated killing of intracellular mycobacteria by macrophages is a P2X(7)-dependent process inducing bacterial death by phagosome-lysosome fusion.

Mycobacterium tuberculosis survives within host macrophages by actively inhibiting phagosome fusion with lysosomes. Treatment of infected macrophages with ATP induces both cell apoptosis and rapid killing of intracellular mycobacteria. The following studies were undertaken to characterize the effector pathway(s) involved. Macrophages were obtained from p47(phox) and inducible NO synthase gene-disrupted mice (which are unable to produce reactive oxygen and nitrogen radicals, respectively) and P2X(7) gene-disrupted mice. RAW murine macrophages transfected with either the natural resistance-associated macrophage protein gene 1 (Nramp1)-resistant or Nramp1-susceptible gene were also used. The cells were infected with bacille Calmette-Guérin (BCG), and intracellular mycobacterial trafficking was analyzed using confocal and electron microscopy. P2X(7) receptor activation was essential for effective ATP-induced mycobacterial killing, as its bactericidal activity was radically diminished in P2X(7)(-/-) macrophages. ATP-mediated killing of BCG within p47(phox-/-), inducible NO synthase(-/-), and Nramp(s) cells was unaffected, demonstrating that none of these mechanisms have a role in the ATP/P2X(7) effector pathway. Following ATP stimulation, BCG-containing phagosomes rapidly coalesce and fuse with lysosomes. Blocking of macrophage phospholipase D activity with butan-1-ol blocked BCG killing, but not macrophage death. ATP stimulates phagosome-lysosome fusion with concomitant mycobacterial death via P2X(7) receptor activation. Macrophage death and mycobacterial killing induced by the ATP/P2X(7) signaling pathway can be uncoupled, and diverge proximal to phospholipase D activation.     

Phagosome dynamics and function

Phagocytosis of microorganisms and other particles is mediated most efficiently by receptors such as Fc-receptors (FcR) and complement-receptors (C3R). Interaction between these receptors and ligands on the particle results in signal transduction events that lead to actin polymerisation and phagosome formation. The phagosome then undergoes a maturation process whereby it transforms into a phagolysosome. Phagosome maturation depends on interactions (fusion events) with early and late endosomes as well as with lysosomes. The fusion processes are regulated by small GTP-binding proteins and other proteins that are also involved in fusion processes in the endocytic pathway. Although most phagocytosed microorganisms are killed in the lysosome, some pathogens have developed survival strategies and are able to live in the harsh conditions in the phagolysosome or interfere with the maturation process and thereby evade destruction by acid hydrolases.