Showing papers on "Phagosome published in 2019"
TL;DR: This Cell Science at a Glance article and accompanying poster detail the divergence of this distinctive mechanism from that of canonical autophagy by comparing and contrasting shared and unique components of each pathway.
Abstract: Classically, canonical autophagy has been considered a survival mechanism initiated in response to nutrient insufficiency. We now understand that autophagy functions in multiple scenarios where it is necessary to maintain homeostasis. Recent evidence has established that a variety of non-canonical functions for autophagy proteins are mechanistically and functionally distinct from autophagy. LC3-associated phagocytosis (LAP) is one such novel function for autophagy proteins and is a contributor to immune regulation and inflammatory responses across various cell and tissue types. Characterized by the conjugation of LC3 family proteins to phagosome membranes, LAP uses a portion of the canonical autophagy machinery, following ligation of surface receptors that recognize a variety of cargos including pathogens, dying cells, soluble ligands and protein aggregates. However, instead of affecting canonical autophagy, manipulation of the LAP pathway in vivo alters immune activation and inflammatory responses. In this Cell Science at a Glance article and the accompanying poster, we detail the divergence of this distinctive mechanism from that of canonical autophagy by comparing and contrasting shared and unique components of each pathway.
163 citations
TL;DR: An aggregation-induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through a dynamic process, notably specific molecular tailoring of the probe by caspase-1 activation in infected macrophage and accumulation of the residue on phagosomes containing bacteria, leading to light-up fluorescent signals.
Abstract: The detection and elimination of intracellular bacteria remain a major challenge. In this work, we report an aggregation-induced emission (AIE) bioprobe that can detect bacterial infection and kill bacteria surviving inside macrophages through a dynamic process, notably specific molecular tailoring of the probe by caspase-1 activation in infected macrophages and accumulation of the residue on phagosomes containing bacteria, leading to light-up fluorescent signals. Moreover, the AIEgen can serve as a photosensitizer for generation of reactive oxygen species (ROS); and the average ROS indicator fluorescent signal intensity per unit area in the bacterial phagosomes is approximately 2.7-fold higher than that in the cytoplasm. This, in turn, induces bacteria killing with high efficiency and minimal cytotoxicity towards macrophages. We envision that this specific light-up bioprobe may provide a new approach for selective and sensitive detection and eradication of intracellular bacterial infections.
74 citations
TL;DR: It is shown that resorption of the phagolysosome after degradation of its contents requires transfer of PI4P and tethering to the ER, both mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector.
Abstract: Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.
71 citations
TL;DR: It is suggested that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis, and interactions between WIPI2 and ATG16L1 were less critical for Autophagy in the brain, which may allow a low level of autophile to overcome neonatal lethality.
Abstract: Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP-/- mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality. Abbreviations: CCD: coiled-coil domain; CYBB/NOX2: cytochrome b-245: beta polypeptide; GPT/ALT: glutamic pyruvic transaminase: soluble; LAP: LC3-associated phagocytosis; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; NOD: nucleotide-binding oligomerization domain; NADPH: nicotinamide adenine dinucleotide phosphate; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing Beclin 1-interacting protein; SLE: systemic lupus erythematosus; SQSTM1/p62: sequestosome 1; TLR: toll-like receptor; TMEM: transmembrane protein; TRIM: tripartite motif-containing protein; UVRAG: UV radiation resistance associated gene; WD: tryptophan-aspartic acid; WIPI: WD 40 repeat domain: phosphoinositide interacting.
69 citations
TL;DR: The primary question the authors wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.
Abstract: Listeriolysin O (LLO) is an essential determinant of Listeria monocytogenes pathogenesis that mediates the escape of L. monocytogenes from host cell vacuoles, thereby allowing replication in the cytosol without causing appreciable cell death. As a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, LLO is unique in that it is secreted by a facultative intracellular pathogen, whereas all other CDCs are produced by pathogens that are largely extracellular. Replacement of LLO with other CDCs results in strains that are extremely cytotoxic and 10,000-fold less virulent in mice. LLO has structural and regulatory features that allow it to function intracellularly without causing cell death, most of which map to a unique N-terminal region of LLO referred to as the proline, glutamic acid, serine, threonine (PEST)-like sequence. Yet, while LLO has unique properties required for its intracellular site of action, extracellular LLO, like other CDCs, affects cells in a myriad of ways. Because all CDCs form pores in cholesterol-containing membranes that lead to rapid Ca2+ influx and K+ efflux, they consequently trigger a wide range of host cell responses, including mitogen-activated protein kinase activation, histone modification, and caspase-1 activation. There is no debate that extracellular LLO, like all other CDCs, can stimulate multiple cellular activities, but the primary question we wish to address in this perspective is whether these activities contribute to L. monocytogenes pathogenesis.
57 citations
TL;DR: This work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.
Abstract: While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages.
57 citations
TL;DR: The generation, kinetics, and toxicity of reactive species generated in phagocytes are discussed, with a focus on the response of macrophages to internalized pathogens and concentrating on Mycobacterium tuberculosis and Trypanosoma cruzi as examples of bacterial and parasitic infection, respectively.
Abstract: The generation of phagosomal cytotoxic reactive species (i.e., free radicals and oxidants) by activated macrophages and neutrophils is a crucial process for the control of intracellular pathogens. The chemical nature of these species, the reactions they are involved in, and the subsequent effects are multifaceted and depend on several host- and pathogen-derived factors that influence their production rates and catabolism inside the phagosome. Pathogens rely on an intricate and synergistic antioxidant armamentarium that ensures their own survival by detoxifying reactive species. In this review, we discuss the generation, kinetics, and toxicity of reactive species generated in phagocytes, with a focus on the response of macrophages to internalized pathogens and concentrating on Mycobacterium tuberculosis and Trypanosoma cruzi as examples of bacterial and parasitic infection, respectively. The ability of pathogens to deal with host-derived reactive species largely depends on the competence of their antioxidant networks at the onset of invasion, which in turn can tilt the balance toward pathogen survival, proliferation, and virulence over redox-dependent control of infection.
55 citations
TL;DR: A fluorescent, DNA-based combination reporter, cHOClate, is introduced, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively and reveals that phagosome–lysosome fusion was essential not only for phagosomal acidification, but also for providing the chloride necessary for myeloperoxidase activity.
Abstract: Phagocytes destroy pathogens by trapping them in a transient organelle called the phagosome, where they are bombarded with reactive oxygen species (ROS) and reactive nitrogen species (RNS). Imaging reactive species within the phagosome would directly reveal the chemical dynamics underlying pathogen destruction. Here we introduce a fluorescent, DNA-based combination reporter, cHOClate, which simultaneously images hypochlorous acid (HOCl) and pH quantitatively. Using cHOClate targeted to phagosomes in live cells, we successfully map phagosomal production of a specific ROS, HOCl, as a function of phagosome maturation. We found that phagosomal acidification was gradual in macrophages and upon completion, HOCl was released in a burst. This revealed that phagosome-lysosome fusion was essential not only for phagosome acidification, but also for providing the chloride necessary for myeloperoxidase activity. This method can be expanded to image several kinds of ROS and RNS and be readily applied to identify how resistant pathogens evade phagosomal killing.
54 citations
TL;DR: An experimental system to separate interaction stages and single macrophage cells infected with C. albicans from uninfected cells is developed and transcriptional variability in the host and fungus is assessed.
Abstract: The outcome of fungal infections depends on interactions with innate immune cells. Within a population of macrophages encountering Candida albicans, there are distinct host-pathogen trajectories; however, little is known about the molecular heterogeneity that governs these fates. Here we developed an experimental system to separate interaction stages and single macrophage cells infected with C. albicans from uninfected cells and assessed transcriptional variability in the host and fungus. Macrophages displayed an initial up-regulation of pathways involved in phagocytosis and proinflammatory response after C. albicans exposure that declined during later time points. Phagocytosed C. albicans shifted expression programs to survive the nutrient poor phagosome and remodeled the cell wall. The transcriptomes of single infected macrophages and phagocytosed C. albicans displayed a tightly coordinated shift in gene expression co-stages and revealed expression bimodality and differential splicing that may drive infection outcome. This work establishes an approach for studying host-pathogen trajectories to resolve heterogeneity in dynamic populations.
54 citations
TL;DR: The differential growth of the bacterium in these different phagocyte populations appears to be linked to host cell metabolism, and tissue-resident alveolar macrophages, which are biased towards an alternatively activated, M2-like phenotype, are more permissive to bacterial growth than monocyte-derived, inflammatory, M1-like interstitial macrophage populations.
Abstract: Mycobacterium tuberculosis has evolved to become the single greatest cause of death from an infectious agent. The pathogen spends most of its infection cycle in its human host within a phagocyte. The bacterium has evolved to block the normal maturation and acidification of its phagosome and resides in a vacuole contiguous with the early endosomal network. Cytokine-mediated activation of the host cell can overcome this blockage, and an array of antimicrobial responses can limit its survival. The survival of M. tuberculosis in its host cell is fueled predominantly by fatty acids and cholesterol. The ability of M. tuberculosis to degrade sterols is an unusual metabolic characteristic that was likely retained from a saprophytic ancestor. Recent results with fluorescent M. tuberculosis reporter strains demonstrate that bacterial survival differs with the host macrophage population. Tissue-resident alveolar macrophages, which are biased towards an alternatively activated, M2-like phenotype, are more permissive to bacterial growth than monocyte-derived, inflammatory, M1-like interstitial macrophages. The differential growth of the bacterium in these different phagocyte populations appears to be linked to host cell metabolism.
50 citations
TL;DR: Advances in current knowledge of the morphology change that neutrophils undergo are discussed, and an important regulated molecular event is identified which may represent an important target for anti-inflammatory therapy.
Abstract: Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role—killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.
TL;DR: The biological function of the terpene nucleoside 1-TbAd is elucidated, which is made abundantly by virulent but not avirulent Mycobacterium tuberculosis strains, and it is demonstrated that 1-tuberculosinyladenosine regulates the pH and function of host macrophage endolysosomes.
Abstract: Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.
TL;DR: The results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
Abstract: Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria.
TL;DR: Phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross‐presentation to generate MHC‐I‐peptide complexes identical to those produced by conventional antigen processing.
Abstract: During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2 -microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing.
TL;DR: This work highlights recent advances in understanding an antimicrobial pathway called LC3-associated phagocytosis (LAP), which combines features of autophagy with phagocytes, and reviews how LAP targets microbial pathogens and strategies pathogens employ to circumvent LAP.
TL;DR: These adaptations of intracellular bacterial pathogens that manipulate conserved cellular pathways, organelles, and cargo to convert the phagosome into a pathogen-containing vacuole (PCV) reflect the outcome of an aggressive and ongoing molecular arms race at the host-pathogen interface.
Abstract: In this article, we explore the unique adaptations of intracellular bacterial pathogens that manipulate conserved cellular pathways, organelles, and cargo to convert the phagosome into a pathogen-containing vacuole (PCV). The phagosome is a degradative organelle that rapidly acidifies as it delivers cargo to the lysosome to destroy microbes and cellular debris. However, to avoid this fate, intracellular bacterial pathogens hijack the key molecular modulators of intracellular traffic: small GTPases, phospholipids, SNAREs, and their associated effectors. Following uptake, pathogens that reside in the phagosome either remain associated with the endocytic pathway or rapidly diverge from the preprogrammed route to the lysosome. Both groups rely on effector-mediated mechanisms to meet the common challenges of intracellular life, such as nutrient acquisition, vacuole expansion, and evasion of the host immune response. Mycobacteria, Salmonella, and Coxiella serve as a lens through which we explore regulators of the canonical endocytic route and pathogens that seek to subvert it. On the other hand, pathogens such as Chlamydia, Legionella, and Brucella disconnect from the canonical endocytic route. This bifurcation is linked to extensive hijacking of the secretory pathway and repurposing of the PCV into specialized compartments that resemble organelles in the secretory network. Finally, each pathogen devises specific strategies to counteract host immune responses, such as autophagy, which aim to destroy these aberrant organelles. Collectively, each unique intracellular niche and the pathogens that construct them reflect the outcome of an aggressive and ongoing molecular arms race at the host-pathogen interface. Improving our understanding of these well-adapted pathogens can help us refine our knowledge of conserved cell biological processes.
TL;DR: This work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.
TL;DR: Evidence supporting the role of Cl− as a signalling effector is reviewed to help in understanding the molecular and metabolic changes seen in pathologies with altered Cl− transport or under physiological conditions.
Abstract: The specific role of the chloride anion (Cl- ) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl- concentration affect diverse cellular functions such as gene and protein expression and activities, post-translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl- also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl- signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl- transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl- as a signalling effector.
TL;DR: It is concluded that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.
Abstract: By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.
TL;DR: This work shows that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages.
Abstract: To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.
TL;DR: It is indicated that a transition of MAB-S strains into Mab-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading.
Abstract: Mycobacterium abscessus complex (MAB) is a rapidly growing mycobacterium(RGM) whose clinical significance as an emerging human pathogen has been increasing worldwide. It has two types of colony morphology, a smooth (S) type, producing high glycopeptidolipid (GPL) content, and a rough (R) type, which produces low levels of GPLs and is associated with increased virulence. However, the mechanism responsible for their difference in virulence is poorly known. By ultrastructural examination of murine macrophages infected, we found that MAB-R strains could replicate more actively in the macrophage phagosome than the S variants and that they could escape into cytosol via phagosomal rupture. The cytosolic access of MAB-R strains via phagosomal rupture led to enhanced Type I interferon (IFN) production and cell death, which resulted in their cell-to-cell spreading. This behavior can provide an additional niche for the survival of MAB-R strains. In addition, we found that their enhancement of cell death mediated cell spreading are dependent on Type I IFN signaling via comparison of wild-type and IFNAR1 knockout mice. In conclusion, our data indicated that a transition of MAB-S strains into MAB-R variants increased their virulence via enhanced Type I IFN production, which led to enhanced survival in infected macrophage via cell death mediated cell-to-cell spreading. This result provides not only a novel insight into the difference in virulence between MAB-R and -S variants but also hints to their treatment strategy.
TL;DR: A novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen was developed and showed that MHCclass II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells.
TL;DR: The acid sphingomyelinase/ceramide system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions and may serve as a novel therapeutic target for treating infections.
Abstract: Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.
TL;DR: These parasites were more resistant to macrophage-dependent killing than the wild type and yielded higher parasitemias and parasite burden in heart tissue of infected mice, underscoring the role of Fe-SODB as a virulence factor for CD.
Abstract: Trypanosoma cruzi, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During T. cruzi invasion to macrophages, superoxide radical (O2•−) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-phox) activation. In this work, T. cruzi cytosolic Fe-SODB overexpressers (pRIBOTEX–Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-phox−/− macrophages, underscoring the role of O2•− in parasite killing. Herein, we studied the entrance of O2•− and its protonated form, perhydroxyl radical [(HO2•); pKa = 4.8], to T. cruzi at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O2•− and HO2• were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O2•− permeation, because inhibition of the macrophage H+-ATPase proton pump significantly decreased O2•− detection in the internalized parasite. Importantly, O2•− detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O2•− or during macrophage infections. Other mechanisms of O2•− entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O2•− detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB–overexpressing parasites, supporting the role of the cytosolic O2•−-catabolizing enzyme as a virulence factor for CD.
TL;DR: The apparent “stretching” of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage are discussed.
Abstract: Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore-forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent "stretching" of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.
TL;DR: This work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells, and offers possible targets for novel antifungal drugs against these opportunistic human pathogens.
Abstract: Mucormycosis is an emerging fungal infection that is often lethal due to the ineffectiveness of current therapies. Here, we have studied the first stage of this infection-the germination of Mucor circinelloides spores inside phagocytic cells-from an integrated transcriptomic and functional perspective. A relevant fungal gene network is remodeled in response to phagocytosis, being enriched in crucial functions to survive and germinate inside the phagosome, such as nutritional adaptation and response to oxidative stress. Correspondingly, the phagocytic cells induced a specific proinflammatory and apoptotic response to the pathogenic strain. Deletion of fungal genes encoding putative transcription factors (atf1, atf2, and gcn4), extracellular proteins (chi1 and pps1), and an aquaporin (aqp1) revealed that these genes perform important roles in survival following phagocytosis, germination inside the phagosome, and virulence in mice. atf1 and atf2 play a major role in these pathogenic processes, since their mutants showed the strongest phenotypes and both genes control a complex gene network of secondarily regulated genes, including chi1 and aqp1 These new insights into the initial phase of mucormycosis define genetic regulators and molecular processes that could serve as pharmacological targets.IMPORTANCE Mucorales are a group of ancient saprophytic fungi that cause neglected infectious diseases collectively known as mucormycoses. The molecular processes underlying the establishment and progression of this disease are largely unknown. Our work presents a transcriptomic study to unveil the Mucor circinelloides genetic network triggered in fungal spores in response to phagocytosis by macrophages and the transcriptional response of the host cells. Functional characterization of differentially expressed fungal genes revealed three transcription factors and three extracellular proteins essential for the fungus to survive and germinate inside the phagosome and to cause disease in mice. Two of the transcription factors, highly similar to activating transcription factors (ATFs), coordinate a complex secondary gene response involved in pathogenesis. The significance of our research is in characterizing the initial stages that lead to evasion of the host innate immune response and, in consequence, the dissemination of the infection. This genetic study offers possible targets for novel antifungal drugs against these opportunistic human pathogens.
TL;DR: The development of a novel method to accurately measure ADCP activity, which is the mechanism by which antibody-opsonized target cells activate the FcγRs on the surface of macrophages to induce phagocytosis, resulting in internalization and degradation of the target cell through phagosome acidification.
TL;DR: An efficient phage transduction protocol is established to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtPB, and/or SapM, and determine the MCV PI pattern, compartment acidification, and phagosomal escape.
Abstract: The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.
TL;DR: This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1) and directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2).
TL;DR: Endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii is investigated, finding that the mature CCV is less acidic than lysosomes and inducing CCV acidification to pH~4.8 causes C.Burnetii lysis, suggesting C. burningetii actively regulates pH of the matureCCV.
Abstract: Upon host cell infection, the obligate intracellular bacterium Coxiella burnetii resides and multiplies within the Coxiella-Containing Vacuole (CCV). The nascent CCV progresses through the endosomal maturation pathway into a phagolysosome, acquiring endosomal and lysosomal markers, as well as acidic pH and active proteases and hydrolases. Approximately 24-48 hours post infection, heterotypic fusion between the CCV and host endosomes/lysosomes leads to CCV expansion and bacterial replication in the mature CCV. Initial CCV acidification is required to activate C. burnetii metabolism and the Type 4B Secretion System (T4BSS), which secretes effector proteins required for CCV maturation. However, we found that the mature CCV is less acidic (pH~5.2) than lysosomes (pH~4.8). Further, inducing CCV acidification to pH~4.8 causes C. burnetii lysis, suggesting C. burnetii actively regulates pH of the mature CCV. Because heterotypic fusion with host endosomes/lysosomes may influence CCV pH, we investigated endosomal maturation in cells infected with wildtype (WT) or T4BSS mutant (ΔdotA) C. burnetii. In WT-infected cells, we observed a significant decrease in proteolytically active, LAMP1-positive endolysosomal vesicles, compared to mock or ΔdotA-infected cells. Using a ratiometric assay to measure endosomal pH, we determined that the average pH of terminal endosomes in WT-infected cells was pH~5.8, compared to pH~4.75 in mock and ΔdotA-infected cells. While endosomes progressively acidified from the periphery (pH~5.5) to the perinuclear area (pH~4.7) in both mock and ΔdotA-infected cells, endosomes did not acidify beyond pH~5.2 in WT-infected cells. Finally, increasing lysosomal biogenesis by overexpressing the transcription factor EB resulted in smaller, more proteolytically active CCVs and a significant decrease in C. burnetii growth, indicating host lysosomes are detrimental to C. burnetii. Overall, our data suggest that C. burnetii inhibits endosomal maturation to reduce the number of proteolytically active lysosomes available for heterotypic fusion with the CCV, possibly as a mechanism to regulate CCV pH.