The Mycobacterium tuberculosis SecA2 system subverts phagosome maturation to promote growth in macrophages

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.     

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.     

The early phagosomal stage of Francisella tularensis determines optimal phagosomal escape and Francisella pathogenicity island protein expression

Francisella tularensis is an intracellular pathogen that can survive and replicate within macrophages. Following phagocytosis and transient interactions with the endocytic pathway, F. tularensis rapidly escapes from its original phagosome into the macrophage cytoplasm, where it eventually replicates. To examine the importance of the nascent phagosome for the Francisella intracellular cycle, we have characterized early trafficking events of the F. tularensis subsp. tularensis strain Schu S4 in a murine bone marrow-derived macrophage model. Here we show that early phagosomes containing Schu S4 transiently interact with early and late endosomes and become acidified before the onset of phagosomal disruption. Inhibition of endosomal acidification with the vacuolar ATPase inhibitor bafilomycin A1 or concanamycin A prior to infection significantly delayed but did not block phagosomal escape and cytosolic replication, indicating that maturation of the early Francisella-containing phagosome (FCP) is important for optimal phagosomal escape and subsequent intracellular growth. Further, Francisella pathogenicity island (FPI) protein expression was induced during early intracellular trafficking events. Although inhibition of endosomal acidification mimicked the early phagosomal escape defects caused by mutation of the FPI-encoded IglCD proteins, it did not inhibit the intracellular induction of FPI proteins, demonstrating that this response is independent of phagosomal pH. Altogether, these results demonstrate that early phagosomal maturation is required for optimal phagosomal escape and that the early FCP provides cues other than intravacuolar pH that determine intracellular induction of FPI proteins.     

Virulent and avirulent strains of Francisella tularensis prevent acidification and maturation of their phagosomes and escape into the cytoplasm in human macrophages

Francisella tularensis, the agent of tularemia, is an intracellular pathogen, but little is known about the compartment in which it resides in human macrophages. We have examined the interaction of a recent virulent clinical isolate of F. tularensis subsp. tularensis and the live vaccine strain with human macrophages by immunoelectron and confocal immunofluorescence microscopy. We assessed the maturation of the F. tularensis phagosome by examining its acquisition of the lysosome-associated membrane glycoproteins (LAMPs) CD63 and LAMP1 and the acid hydrolase cathepsin D. Two to four hours after infection, vacuoles containing live F. tularensis cells acquired abundant staining for LAMPs but little or no staining for cathepsin D. However, after 4 h, the colocalization of LAMPs with live F. tularensis organisms declined dramatically. In contrast, vacuoles containing formalin-killed bacteria exhibited intense staining for all of these late endosomal/lysosomal markers at all time points examined (1 to 16 h). We examined the pH of the vacuoles 3 to 4 h after infection by quantitative immunogold staining and by fluorescence staining for lysosomotropic agents. Whereas phagosomes containing killed bacteria stained intensely for these agents, indicating a marked acidification of the phagosomes (pH 5.5), phagosomes containing live F. tularensis did not concentrate these markers and thus were not appreciably acidified (pH 6.7). An ultrastructural analysis of the F. tularensis compartment revealed that during the first 4 h after uptake, the majority of F. tularensis bacteria reside within phagosomes with identifiable membranes. The cytoplasmic side of the membranes of approximately 50% of these phagosomes was coated with densely staining fibrils of approximately 30 nm in length. In many cases, these coated phagosomal membranes appeared to bud, vesiculate, and fragment. By 8 h after infection, the majority of live F. tularensis bacteria lacked any ultrastructurally discernible membrane separating them from the host cell cytoplasm. These results indicate that F. tularensis initially enters a nonacidified phagosome with LAMPs but without cathepsin D and that the phagosomal membrane subsequently becomes morphologically disrupted, allowing the bacteria to gain direct access to the macrophagic cytoplasm. The capacity of F. tularensis to alter the maturation of its phagosome and to enter the cytoplasm is likely an important element of its capacity to parasitize macrophages and has major implications for vaccine development.     

Proteomic analysis of Legionella-containing phagosomes isolated from Dictyostelium

Legionella pneumophila, the agent of Legionnaires’ disease, replicates intracellularly within specialized phagosomes of human macrophages and amoebae. In this study, we have developed a protocol for the isolation of Legionella-containing phagosomes from Dictyostelium discoideum. Cell fractionation, two-dimensional gel electrophoresis and MALDI-TOF MS combined with genomic data identified 157 phagosome host proteins. In addition to proteins with an evident role in phagosome maturation, we identified proteins for which a function remains to be elucidated. Possible interactions of coronin with cytosolic NADPH oxidase components and protein kinase C inhibitors which together may lead to an inhibition of phagosomal superoxide generation are discussed. Comparative proteomics of phagosomes containing highly virulent L. pneumophila Corby versus less virulent L. hackeliae revealed distinctive protein expression patterns, e.g., an abundance of RhoGDI in L. hackeliae degrading phagosomes versus little RhoGDI in L. pneumophila Corby replicative phagosomes. We present a kinetic dissection of phagosome maturation including the complex alterations of the phagosome protein composition. A reference flow chart suggests so far unrecognized consequences of infection for host cell physiology, actin degradation on phagosomes, and a putative cysteine proteinase inhibitor interference with lysosomal enzyme sorting and activation processes.     

How nascent phagosomes mature to become phagolysosomes

Phagocytosis mediates the clearance of apoptotic bodies and also the elimination of microbial pathogens. The nascent phagocytic vacuole formed upon particle engulfment lacks microbicidal and degradative activity. These capabilities are acquired as the phagosome undergoes maturation; a progressive remodeling of its membrane and contents that culminates in the formation of phagolysosomes. Maturation entails orderly sequential fusion of the phagosomal vacuole with specialized endocytic and secretory compartments. Concomitantly, the phagosomal membrane undergoes both inward and outward vesiculation and tubulation followed by fission, thereby recycling components and maintaining its overall size. Here, we summarize what is known about the molecular machinery that governs this complex metamorphosis of phagosome maturation.     

The IRG proteins: a function in search of a mechanism

The IRG proteins (p47 GTPases) constitute one of the strongest resistance systems known to be active against intracellular pathogens in mice. The proteins are induced by interferons and assemble on phagosomes and parasitophorous vacuoles of a number of different micro-organisms in all cell types assayed. There are presently three experimentally based views as to how they exert their cell-autonomous activity against intracellular pathogens: blocking of interferon-mediated acceleration of phagosome maturation, induction of autophagic membranes, and direct destruction of the parasitophorous vacuole membrane. Failure of hemopoietic stem cells during infection is associated with targeted deletion of one IRG protein, Irgm1. The significance of this non-cell-autonomous phenotype is discussed.     

Vacuolar ATPase in phago(lyso)some biology

Many eukaryotic cells ingest extracellular particles in a process termed phagocytosis which entails the generation of a new intracellular compartment, the phagosome. Phagosomes change their composition over time and this maturation process culminates in their fusion with acidic, hydrolase-rich lysosomes. During the maturation process, degradation and, when applicable, killing of the cargo may ensue. Many of the events that are pathologically relevant depend on strong acidification of phagosomes by the ‘vacuolar’ ATPase (V-ATPase). This protein complex acidifies the lumen of some intracellular compartments at the expense of ATP hydrolysis. We discuss here the roles and importance of V-ATPase in intracellular trafficking, its distribution, inhibition and activities, its role in the defense against microorganisms and the counteractivities of pathogens.     

Inside the phagosome: A bacterial perspective

The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes.     

Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival.     

Stable accumulation of p67phox at the phagosomal membrane and ROS production within the phagosome

Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67(phox) during phagocytosis by videomicroscopy. p67(phox) is involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67(phox)-citrine, expressed in myeloid PLB-985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67(phox)-citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67(phox)-citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67(phox) is stable in this complex. Furthermore, the time of the presence of p67(phox)-citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67(phox)-citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67(phox) for ROS production on the level of individual phagosomes.     

Phagosome maturation proceeds independently of stimulation of toll-like receptors 2 and 4

Toll-like receptors modulate many aspects of the innate immune response. Recent reports suggest that the maturation of phagosomes following particle uptake is modulated through signaling of Toll-like receptors. In the current study, the kinetics of phagosome maturation was evaluated quantitatively by ratio fluorometry to determine the lumenal pH of the phagosomes and a FRET-based technique to determine the degree of phagosome/lysosome fusion. Profiles generated for phagosomes containing experimental particles with or without the TLR ligands Pam3Cys-Ser-(Lys)4 or LPS failed to reveal a difference in maturation despite activating TLR-signaling pathways. Moreover, while macrophages defective in individual TLRs generated phagosome maturation profiles identical to wild-type macrophages, MyD88-deficient macrophages exhibited a marked depression in phagosome/lysosome fusion that appears independent of short-term TLR-mediated effects. The results demonstrate that the rate of maturation of phagosomes proceeds independently of TLR signaling pathways.     

Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications

Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments.     

SNX3 recruits to phagosomes and negatively regulates phagocytosis in dendritic cells

Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria‐derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well‐regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol‐3,4,5‐triphosphate [PI(3,4,5)P₃] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P₃ is accompanied by high levels of phosphatidylinositol‐3‐phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox‐homology (PX) domain‐containing cytoplasmic and membrane‐associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid‐binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid‐binding protein, early endosome antigen‐1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid‐binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes.     

Phagocytic receptors dictate phagosomal escape and intracellular proliferation of Francisella tularensis

Francisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Various in vitro models of Francisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent of Francisella phagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strain Francisella tularensis subsp. tularensis Schu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the early Francisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate of Francisella by delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.     

Vacuole acidification is not required for survival of Salmonella enterica serovar typhimurium within cultured macrophages and epithelial cells

Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellular Salmonella enterica serovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential for Salmonella intracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.     

Redistribution of ecto-5'-nucleotidase during phagocytosis by guinea pig polymorphonuclear leukocytes

We demonstrate that 5'-nucleotidase (5'NT), an ectoenzyme of guinea pig polymorphonuclear leukocytes, is largely excluded from phagosomal membrane, rather than internalized randomly during phagocytosis of heat-killed bacteria, latex microbeads, or zymosan particles. Cells were fixed in 0.25% glutaraldehyde (pH 6.3) at 4 degrees C for 10 min and incubated in a cytochemical medium for the demonstration of 5'NT. In the nonphagocytosing cells, 5'NT was evenly distributed on the external side of the plasma membrane. In cells phagocytosing bacteria, 5'NT appeared to be cleared from the nascent phagosomal membrane; after 5 min of phagocytosis, most of the phagocytic vacuoles containing bacteria, latex, or zymosan particles were devoid of reaction product. When phagosomes containing latex particles were isolated and biochemically assayed, they contained less than 3% of the total cellular 5'NT activity even after 60 min of phagocytosis, and at that time the total cellular 5'NT activity had not declined. When the diazonium salt of sulfanilic acid (DASA), a nonpermeable ectoenzyme inhibitor, was used to determine the distribution of extracellular and intracellular 5'NT activity, no increase in DASA-insensitive intracellular 5'NT was found after phagocytosis of latex or opsonized zymosan. Cytochemical and biochemical evidence led us to conclude that 5'NT is excluded from phagosomal membrane, and that the exclusion is due to redistribution rather than to inactivation by granule enzymes.     

LC3-associated phagocytosis in microbial pathogenesis

Phagocytosis is essential for uptake and elimination of pathogenic microorganisms. Autophagy is a highly conserved mechanism for incorporation of cellular constituents to replenish nutrients by degradation. Recently, parts of the autophagy machinery – above all microtubule-associated protein 1 light chain 3 (LC3) – were found to be specifically recruited to phagosomal membranes resulting in phagosome-lysosome fusion and efficient degradation of internalized cargo in a process termed LC3-associated phagocytosis (LAP). Many pathogenic bacterial, fungal and parasitic microorganisms reside within LAP-targeted single-membrane phagosomes or vacuoles after infection of host cells. In this minireview we describe the state of knowledge on the interaction of pathogens with LAP or LAP-like pathways and report on various pathogens that have evolved strategies to circumvent degradation in LAP compartments.     

Leishmania donovani requires functional Cdc42 and Rac1 to prevent phagosomal maturation

Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase C alpha. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.