The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination.
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 opsonisation 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 internalised 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.
Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.
To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb infected (single infected). The controlled Mtb infection was disrupted upon mTOR inhibition resulting in increased Mtb replication in a dose-dependent manner which was more pronounced during co-infection. The increased Mtb replication could be explained by the marked reduction in phagosome acidification upon mTOR inhibition. Autophagy stimulation targeting mTORC1 clearly induced a basal autophagy with flux that was unlinked to the subcellular environment of the Mtb vacuoles, which showed a concurrent suppression in acidification and maturation/flux. Overall our findings indicate that mTOR inhibition during Mtb or HIV/Mtb co-infection interferes with phagosomal maturation, thereby supporting mycobacterial growth during low-dose and controlled infection. Therefore pharmacological induction of autophagy through targeting of the canonical mTORC1-pathway should be handled with caution during controlled tuberculosis, since this could have serious consequences for patients with HIV/Mtb co-infection.
Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.
The ability to suppress host macrophage apoptosis is essential for M. tuberculosis (Mtb) to replicate intracellularly while protecting it from antibiotic treatment. We recently described that Mtb infection upregulated expression of the host phosphatase PPM1A, which impairs the antibacterial response of macrophages. Here we establish PPM1A as a checkpoint target used by Mtb to suppress macrophage apoptosis. Overproduction of PPM1A suppressed apoptosis of Mtb-infected macrophages by a mechanism that involves inactivation of the c-Jun N-terminal kinase (JNK). Targeted depletion of PPM1A by shRNA or inhibition of PPM1A activity by sanguinarine restored JNK activation, resulting in increased apoptosis of Mtb-infected macrophages. We also demonstrate that activation of JNK by subtoxic concentrations of anisomycin induced selective apoptotic killing of Mtb-infected human macrophages, which was completely blocked in the presence of a specific JNK inhibitor. Finally, selective killing of Mtb-infected macrophages and subsequent bacterial release enabled rifampicin to effectively kill Mtb at concentrations that were insufficient to act against intracellular Mtb, providing proof of principle for the efficacy of a “release and kill” strategy. Taken together, these findings suggest that drug-induced selective apoptosis of Mtb-infected macrophages is achievable.
The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery.
Macrophages internalize pathogens for intracellular degradation. An important part of this process is the phagosomal transport from the cell periphery to the perinuclear region. Biochemical factors are known to influence the fate of phagosomes. Here, we show that the size of phagosomes also has a strong influence on their transport. We found that large phagosomes are transported persistently to the nucleus, whereas small phagosomes show strong bidirectional transport. We show that dynein motors play a larger role in the transport of large phagosomes, whereas actin filament-based motility plays a larger role in the transport of small phagosomes. Furthermore, we investigated the spatial distribution of dyneins and microtubules around phagosomes and hypothesize that dynein and microtubule density differences between the nucleus-facing side of phagosomes and the opposite side could explain part of the observed transport characteristics. Our findings suggest that a size-dependent cellular sorting mechanism might exist that supports macrophages in their immunological roles.
Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) integrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor-differentiated macrophages more than in granulocyte-macrophage colony-stimulating factor-differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial replication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host.
Mycobacterium tuberculosis (M.tb) has evolved mechanisms to evade its destruction in phagolysosomes, where it successfully survives and replicates within phagocytes. Recent studies have shown that virulent strains of M.tb can translocate from the phagosome into the cytosol of dendritic cells (DC). The molecular mechanisms by which virulent M.tb strains can escape the phagosome remain unknown. Here we show that the virulent M.tb strain H37Rv, but not the vaccine strain Bacille Calmette-Guérin (BCG), escapes from the phagolysosome and enters the cytosol by interfering with the TLR-2-MyD88 signaling pathway. Using H37Rv mutants, we further demonstrate that the region of difference-1 (RD-1) locus and ESAT-6, a gene within the RD-1 locus, play an important role in the capacity of M.tb to migrate from the phagosome to the cytosol of macrophages. H37Rv, BCG, H37RvΔRD1, and H37RvΔESAT6 were able to translocate to the cytosol in macrophages derived from TLR-2- and MyD88-deficient animals, whereas only virulent H37Rv was able to enter the cytosol in macrophages from wild type mice. Therefore, signaling through the TLR-2-MyD88 pathway in macrophages plays an important role in confining M.tb within phagolysomes. Virulent strains of M.tb have evolved mechanisms to subvert this pathway, thus facilitating their translocation to the cytosol and to escape the toxic microenvironment of the phagosome or phagolysosome.