Atg5-Independent Sequestration of Ubiquitinated Mycobacteria

Like several other intracellular pathogens, Mycobacterium marinum (Mm) escapes from phagosomes into the host cytosol where it can polymerize actin, leading to motility that promotes spread to neighboring cells. However, only ∼25% of internalized Mm form actin tails, and the fate of the remaining bacteria has been unknown. Here we show that cytosolic access results in a new and intricate host pathogen interaction: host macrophages ubiquitinate Mm, while Mm shed their ubiquitinated cell walls. Phagosomal escape and ubiquitination of Mm occured rapidly, prior to 3.5 hours post infection; at the same time, ubiquitinated Mm cell wall material mixed with host-derived dense membrane networks appeared in close proximity to cytosolic bacteria, suggesting cell wall shedding and association with remnants of the lysed phagosome. At 24 hours post-infection, Mm that polymerized actin were not ubiquitinated, whereas ubiquitinated Mm were found within LAMP-1–positive vacuoles resembling lysosomes. Though double membranes were observed which sequestered Mm away from the cytosol, targeting of Mm to the LAMP-1–positive vacuoles was independent of classical autophagy, as demonstrated by absence of LC3 association and by Atg5-independence of their formation. Further, ubiquitination and LAMP-1 association did not occur with mutant avirulent Mm lacking ESX-1 (type VII) secretion, which fail to escape the primary phagosome; apart from its function in phagosome escape, ESX-1 was not directly required for Mm ubiquitination in macrophages or in vitro. These data suggest that virulent Mm follow two distinct paths in the cytosol of infected host cells: bacterial ubiquitination is followed by sequestration into lysosome-like organelles via an autophagy-independent pathway, while cell wall shedding may allow escape from this fate to permit continued residence in the cytosol and formation of actin tails.     

Expulsion of live pathogenic yeast by macrophages

Phagocytic cells, such as neutrophils and macrophages, perform a critical role in protecting organisms from infection by engulfing and destroying invading microbes . Although some bacteria and fungi have evolved strategies to survive within a phagocyte after uptake, most of these pathogens must eventually kill the host cell if they are to escape and infect other tissues . However, we now demonstrate that the human fungal pathogen Cryptococcus neoformans is able to escape from within macrophages without killing the host cell by a novel expulsive mechanism. This process occurs in both murine J774 cells and primary human macrophages. It is extremely rapid and yet can occur many hours after phagocytosis of the pathogen. Expulsion occurs independently of the initial route of phagocytic uptake and does not require phagosome maturation . After the expulsive event, both the host macrophage and the expelled C. neoformans appear morphologically normal and continue to proliferate, suggesting that this process may represent an important mechanism by which pathogens are able to escape from phagocytic cells without triggering host cell death and thus inflammation .     

Anthrax: early steps of the intracellular stage of infection development

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.     

Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV-related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein-protein interactions. Specific interactions between different Icm proteins were detected by yeast two-hybrid and gel overlay analysis. These data support a model in which the IcmQ-IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS-IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.     

Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen

Cryptococcus is a potentially fatal fungal pathogen and a leading cause of death in immunocompromised patients. As an opportunistic and facultative intracellular pathogen of humans, Cryptococcus exhibits a complex set of interactions with the host immune system in general, and macrophages in particular. Cryptococcus is resistant to phagocytosis but is also able to survive and proliferate within the mature phagolysosome. It can cause the lysis of host cells, can be transferred between macrophages or exit non‐lytically via vomocytosis. Efficient phagocytosis is reliant on opsonization and Cryptococcus has a number of anti‐phagocytic strategies including formation of titan cells and a thick polysaccharide capsule. Following uptake, phagosome maturation appears to occur normally, but the internalized pathogen is able to survive and replicate. Here we review the interactions and host manipulation processes that occur within cryptococcal‐infected macrophages and highlight areas for future research.     

Evidence for pore-forming ability by Legionella pneumophila

Legionella pneumophila is the cause of Legionnaires' pneumonia. After internalization by macrophages, it bypasses the normal endocytic pathway and occupies a replicative phagosome bound by endoplasmic reticulum. Here, we show that lysis of macrophages and red blood cells by L . pneumophila was dependent on dotA and other loci known to be required for proper targeting of the phagosome and replication within the host cell. Cytotoxicity occurred rapidly during a high-multiplicity infection, required close association of the bacteria with the eukaryotic cell and was a form of necrotic cell death accompanied by osmotic lysis. The differential cytoprotective ability of high-molecular-weight polyethylene glycols suggested that osmotic lysis resulted from insertion of a pore less than 3 nm in diameter into the plasma membrane. Results concerning the uptake of membrane-impermeant fluorescent compounds of various sizes are consistent with the osmoprotection analysis. Therefore, kinetic and genetic evidence suggested that the apparent ability of L . pneumophila to insert a pore into eukaryotic membranes on initial contact may play a role in altering endocytic trafficking events within the host cell and in the establishment of a replicative vacuole.     

Role of Immune Serum in the Killing of Helicobacter pylori by Macrophages

Background: Helicobacter pylori infection can lead to the development of gastritis, peptic ulcers and gastric cancer, which makes this bacterium an important concern for human health. Despite evoking a strong immune response in the host, H. pylori persists, requiring complex antibiotic therapy for eradication. Here we have studied the impact of a patient’s immune serum on H. pylori in relation to macrophage uptake, phagosome maturation, and bacterial killing. Materials and Methods: Primary human macrophages were infected in vitro with both immune serum‐treated and control H. pylori. The ability of primary human macrophages to kill H. pylori was characterized at various time points after infection. H. pylori phagosome maturation was analyzed by confocal immune fluorescence microscopy using markers specific for H. pylori, early endosomes (EEA1), late endosomes (CD63) and lysosomes (LAMP‐1). Results: Immune serum enhanced H. pylori uptake into macrophages when compared to control bacteria. However, a sufficient inoculum remained for recovery of viable H. pylori from macrophages, at 8 hours after infection, for both the serum‐treated and control groups. Both serum‐treated and control H. pylori phagosomes acquired EEA1 (15 minutes), CD63 and LAMP‐1 (30 minutes). These markers were then retained for the rest of an 8 hour time course. Conclusions: While immune sera appeared to have a slight positive effect on bacterial uptake, both serum‐treated and control H. pylori were not eliminated by macrophages. Furthermore, the same disruptions to phagosome maturation were observed for both serum‐treated and control H. pylori. We conclude that to eliminate H. pylori, a strategy is required to restore the normal process of phagosome maturation and enable effective macrophage killing of H. pylori, following a host immune response.     

Regulation of Legionella phagosome maturation and infection through flagellin and host Ipaf

Legionella pneumophila is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the Legionella-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in Legionella degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular Legionella replication. However, the mechanism by which Ipaf restricts Legionella replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for Legionella to activate caspase-1 in macrophages. Activation of caspase-1 in response to Legionella infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed Legionella replication. Accordingly a Legionella mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. In vivo studies confirmed that flagellin and Ipaf play an important role in the control of Legionella clearance. These results reveal that Ipaf restricts Legionella replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.     

Temporal and spatial trigger of post‐exponential virulence‐associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol

During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila‐containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post‐exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two‐component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy‐based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two‐component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore‐forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence‐associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol.     

The relationship between the fate of the agent of toxoplasmosis, Toxoplasma gondii, in macrophages cultivated in vitro and the virulence of the parasites]

Comparative light microscopic and electron microscopic studies of development of a highly-virulent RH strain and a less virulent Lagrave strain of Toxoplasma cultivated in macrophages in vitro were made. Contrary to active multiplication of the highly virulent strain most of toxoplasmas of a less virulent strain disintegrated the first hours, degenerating completely in 24-48 hours after the penetration into the macrophages. Submicroscopic study showed no marked cytological changes of macrophages infected with a less virulent strain in comparison with the marked changes of the nuclei in macrophages infected with the RH strain. Disintegration of parasites within the phagosome was accompanied by a gradual transformation of an originally double (or triple) vacuolar membrane to a single membrane and by the disappearance of an additional layer of mitochondria and endoplasmic reticulum elements surrounding the vacuolar membrane. It is likely that the capacity of toxoplasma to develop in macrophages in vitro could become an additional marker to its virulence for albino mice.     

Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast-form fungus which causes pulmonary and meningeal infections preferentially in the immunocompromised host. It is thought that cell-mediated immunity is important for acquired resistance against cryptococcosis with activated macrophages as the final effector cells. However, specific polysaccharides in the capsule of C. neoformans protect the fungus from adherence to phagocytes and from subsequent phagocytosis. We have studied extracellular killing of C. neoformans by IFN-gamma-activated macrophages and their products. Murine bone marrow-derived macrophages stimulated with rIFN-gamma for 24 h were able to effectively suppress the growth of C. neoformans and the effect of IFN-gamma was augmented by LPS. Killing of C. neoformans was also achieved by cell-free supernatants from bone marrow-derived macrophages stimulated with IFN-gamma plus LPS. Our results indicate that killing of C. neoformans by activated macrophages is independent from toxic oxygen radicals and mediated by secreted protein(s) of apparent molecular mass of 15 and 30 kDa. These findings indicate that activated macrophages play a major role in host defense, although the fungus resists phagocytosis and remains in the extracellular milieu.     

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.     

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.     

Entry and survival of pathogenic mycobacteria in macrophages

Pathogenic mycobacteria, including Mycobacterium tuberculosis, are phagocytosed by macrophages but manage to survive within the mycobacterial phagosome. Recent work has shed some more light on the mechanisms of mycobacterial entry and survival inside macrophages. Two host cell components, the steroid cholesterol and a phagosomal coat protein termed TACO were found to play crucial roles in the establishment of an intracellular infection. This review describes how these findings may help to understand the circumvention of the normal trafficking routes inside host cells by mycobacteria.     

The role of macrophage cell death in tuberculosis

Studies of host responses to infection have traditionally focused on the direct antimicrobial activity of effector molecules (antibodies, complement, defensins, reactive oxygen and nitrogen intermediates) and immunocytes (macrophages, lymphocytes, and neutrophils among others). The discovery of the systems for programmed cell death of eukaryotic cells has revealed a unique role for this process in the complex interplay between microorganisms and their cellular targets or responding immunocytes. In particular, cells of the monocyte/macrophage lineage have been demonstrated to undergo apoptosis following intracellular infection with certain pathogens that are otherwise capable of surviving within the hostile environment of the phagosome or which can escape the phagosome. Mycobacterium tuberculosis is a prototypical 'intracellular parasite' of macrophages, and the direct induction of macrophage apoptosis by this organism has recently been reported from several laboratories. This paper reviews the current understanding of the mechanism and regulation of macrophage apoptosis in response to M. tuberculosis and examines the role this process plays in protective immunity and microbial virulence.     

Lysosomal ubiquitin and the demise of Mycobacterium tuberculosis

The antimicrobial activity of macrophages is mediated by both oxidative and non-oxidative mechanisms. Oxidative mechanisms include the action of reactive oxygen and nitrogen intermediates on bacteria. Non-oxidative mechanisms include the maturation of the phagosome into an acidified, hydrolytically active compartment as well as the action of antimicrobial peptides. Mycobacterium tuberculosis parasitizes the host macrophage by arresting the normal maturation of its phagosome and resides in a compartment that fails to fuse with lysosomes. When bacteria are unable to regulate phagosome maturation, such as in activated macrophages, they are delivered to lysosomal compartments, where they are killed. Recent data indicate that the antimycobacterial mechanism of the lysosome is due in part to the action of ubiquitin-derived peptides.     

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

The natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behaviour during early stages of the infection of Dictyostelium discoideum amoebae. Bacteria were labelled with a red fluorescent marker, and selected proteins and organelles in the host were labelled with GFP, allowing the dynamics and interactions of L. pneumophila -containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H(+)-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host.     

Drosophila S2 cells: an alternative infection model for Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that infects humans and animals. Its pathogenic strategy involves the expression of virulence proteins that mediate intracytosolic growth and cell-to-cell spread. A key virulence protein is the cholesterol-dependent cytolysin, listeriolysin O (LLO), which is largely responsible for mediating escape from the phagosome into the host cytosol. To study further the host processes exploited during L. monocytogenes infection, we sought to develop Drosophila S2 cells as a model for infection. Here, we show that S2 cells share a number of properties with mammalian cell culture models of infection. As with mouse macrophages, LLO was required for phagosomal escape from S2 cells. Furthermore, vacuolar escape was dependent on their acidification via the ATPase proton pumps, as bafilomycin A1 treatment sharply decreased escape. However, unlike in mouse macrophages, LLO mutants replicated in the phagosome of S2 cells. Drosophila cells are cholesterol auxotrophs, and exogenous cholesterol increased the infection rate of L. monocytogenes (LLO independent) and also augmented the efficiency of vacuolar escape (LLO dependent). With available genetic tools such as RNA interference, S2 cells could become an important model in the study of host-pathogen interactions.     

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.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.