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Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.
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The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.
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The understanding of the inactivation process of ingested bacteria by phagocytes is a key focus in the field of host-pathogen interactions. Dictyostelium is a model organism that has been at the forefront of uncovering the mechanisms underlying this type of interaction. In this study, we describe an assay designed to measure the inactivation of Klebsiella aerogenes in the phagosomes of Dictyostelium discoideum.
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Dictyostelium , Dictyostelium/microbiología , Dictyostelium/fisiología , Interacciones Huésped-Patógeno , Fagosomas/microbiología , Fagosomas/metabolismo , FagocitosisRESUMEN
The molecular pathology of lung injury in patients with Corona Virus Disease 2019 (COVID-19) remain unclear. In this study, we performed a proteomic study of lung tissues from seven patients with COVID-19, and eight without. Lung parenchymal tissues with COVID-19 were obtained from autopsy samples, while control samples were obtained from paracancerous tissues. Proteins were extracted using phenol extraction. A tandem mass tag-based quantitative proteomic approach combined with bioinformatic analysis was used to detect proteomic changes in the SARS-CoV-2-infected lung tissues. A total of 6,602, and 6,549 proteins were identified in replicates 1 and 2, respectively. Of these, 307, and 278, respectively, were identified as differentially expressed proteins (DEPs). In total, 216 DEPs were identified in this study. These proteins were enriched in 189 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The downregulated proteins are mainly involved in focal adhesion (n = 5), and the PI3K-Akt signaling pathway (n = 4). The upregulated proteins were related to neutrophil extracellular trap (NET) formation (n = 16), and the phagosome pathway (n = 11). The upregulated proteins in these two pathways interact with one another. Further immunohistochemistry verified NET enrichment in the tissues with COVID-19 compared to the controls. Our results systematically outlined the proteomic profiles of the lung's response to SARS-CoV-2 infection and indicated that NET formation was hyper-activated. These results will hopefully provide new evidence for understanding the mechanism behind fatal COVID-19.
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Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species.Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.
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The humoral arm of mammalian innate immunity regulates several molecular mechanisms involved in resistance to pathogens, inflammation, and tissue repair. Recent studies highlight the crucial role played by humoral mediators in granulomatous inflammation. However the molecular mechanisms linking the function of these soluble molecules to the initiation and maintenance of granulomas remain elusive. We propose that humoral innate immunity coordinates fundamental physiological processes in macrophages which, in turn, initiate activation and transformation events that enable granuloma formation. We discuss the involvement of humoral mediators in processes such as immune activation, phagocytosis, metabolism, and tissue remodeling, and how these can dictate macrophage functionality during granuloma formation. These advances present opportunities for discovering novel disease factors and developing targeted, more effective treatments for granulomatous diseases.
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Granuloma , Inmunidad Humoral , Inmunidad Innata , Macrófagos , Humanos , Animales , Granuloma/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Inflamación/inmunología , Transducción de Señal/inmunologíaRESUMEN
Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.
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Señalización del Calcio , Calcio , Neutrófilos , Fagocitosis , Fagosomas , Humanos , Calcio/metabolismo , Fagosomas/metabolismo , Neutrófilos/metabolismo , Citosol/metabolismo , Membrana Celular/metabolismoRESUMEN
Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.
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Células Epiteliales , Mastitis Bovina , Fagocitosis , Infecciones Estafilocócicas , Staphylococcus aureus , Proteínas de Unión al GTP rab , Animales , Bovinos , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Staphylococcus aureus/fisiología , Femenino , Células Epiteliales/microbiología , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/microbiología , Mastitis Bovina/microbiología , Glándulas Mamarias Animales/microbiología , Endosomas/metabolismo , Endosomas/microbiología , Lisosomas/metabolismo , Lisosomas/microbiología , Línea Celular , Fagosomas/microbiologíaRESUMEN
Edwardsiella tarda (Et) is a zoonotic gram-negative pathogen with a diverse host range, including fish. However, the in-depth molecular mechanisms underlying the response of Labeo rohita (rohu) kidney to Et are poorly understood. A proteomic and histopathological analysis was performed for the rohu kidney after Et infection. The histopathology of the infected rohu kidney showed vacuolation and necrosis. After LC-MS/MS analysis, ~1240 proteins were identified with ≥2 unique peptides. A total of 96 differentially abundant proteins (DAPs) were observed between the control and Et infected group (ET). Metascape and STRING analysis were used for the gene ontology (GO), and protein-protein interaction network (PPI) for the significant pathways of DAPs. In PPI, low-abundant proteins were mapped to metabolic pathways and oxidative phosphorylation (cox5ab, uqcrfs1). High-abundance proteins were mapped to ribosomes (rplp2), protein process in the ER (hspa8), and immune system (ptgdsb.1, muc2). Our label-free proteomic approach in the rohu kidney revealed abundant enriched proteins involved in vesicle coat (ehd4), complement activation (c3a.1, c9, c7a), phagosome (thbs4, mapk1), metabolic reprogramming (hao1, glud1a), wound healing (vim, alox5), and the immune system (psap) after Et infection. A targeted proteomics approach of multiple reaction monitoring (MRM) validated the DAPs (nprl3, ambp, vmo1a, hspg2, muc2, hao1 and glud1a) between control and ET. Overall, the current analysis of histology and proteome in the rohu kidney provides comprehensive data on pathogenicity and the potential immune proteins against Et.
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Edwardsiella tarda , Infecciones por Enterobacteriaceae , Enfermedades de los Peces , Proteínas de Peces , Riñón , Proteómica , Animales , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/metabolismo , Infecciones por Enterobacteriaceae/microbiología , Riñón/microbiología , Riñón/metabolismo , Proteínas de Peces/metabolismo , Cyprinidae/metabolismo , Cyprinidae/microbiología , Proteoma/análisis , Mapas de Interacción de Proteínas , Espectrometría de Masas en TándemRESUMEN
Pathogenic Rhodococcus equi release the virulence-associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH-specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m-1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono- and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA-mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes.
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Rhodococcus equi , Proteína Estafilocócica A , Virulencia , Proteína Estafilocócica A/metabolismo , Factores de Virulencia/metabolismo , Rhodococcus equi/metabolismo , Proteínas Bacterianas/metabolismo , LípidosRESUMEN
Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.
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Macrófagos , Rhinovirus , Humanos , Macrófagos/microbiología , Macrófagos Alveolares , Fagocitosis , BacteriasRESUMEN
Drug-resistant tuberculosis (TB) outbreak has emerged as a global public health crisis. Therefore, new and innovative therapeutic options like host-directed therapies (HDTs) through novel modulators are urgently required to overcome the challenges associated with TB. In the present study, we have investigated the anti-mycobacterial effect of 4-(Benzyloxy)phenol. Cell-viability assay asserted that 50⯵M of 4-(Benzyloxy)phenol was not cytotoxic to phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. It was observed that 4-(Benzyloxy)phenol activates p53 expression by hindering its association with KDM1A. Increased ROS, intracellular Ca2+ and phagosome-lysosome fusion, were also observed upon 4-(Benzyloxy)phenol treatment. 4-(Benzyloxy)phenol mediated killing of intracellular mycobacteria was abrogated in the presence of specific inhibitors of ROS, Ca2+ and phagosome-lysosome fusion like NAC, BAPTA-AM, and W7, respectively. We further demonstrate that 4-(Benzyloxy)phenol mediated enhanced ROS production is mediated by acetylation of p53. Blocking of p53 acetylation by Pifithrin-α (PFT- α) enhanced intracellular mycobacterial growth by blocking the mycobactericidal effect of 4-(Benzyloxy)phenol. Altogether, the results showed that 4-(Benzyloxy)phenol executed its anti-mycobacterial effect by modulating p53-mediated ROS production to regulate phagosome-lysosome fusion through Ca2+ production.
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Mycobacterium , Proteína p53 Supresora de Tumor , Humanos , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/farmacología , Macrófagos , Fenol , Células THP-1 , Fagosomas/metabolismo , Fagosomas/microbiología , Lisosomas/metabolismo , Mycobacterium/metabolismo , Fenoles/farmacología , Fenoles/metabolismoRESUMEN
In the tug-of-war between host and pathogen, both evolve to combat each other's defence arsenals. Intracellular phagosomal bacteria have developed strategies to modify the vacuolar niche to suit their requirements best. Conversely, the host tries to target the pathogen-containing vacuoles towards the degradative pathways. The host cells use a robust system through intracellular trafficking to maintain homeostasis inside the cellular milieu. In parallel, intracellular bacterial pathogens have coevolved with the host to harbour strategies to manipulate cellular pathways, organelles, and cargoes, facilitating the conversion of the phagosome into a modified pathogen-containing vacuole (PCV). Key molecular regulators of intracellular traffic, such as changes in the organelle (phospholipid) composition, recruitment of small GTPases and associated effectors, soluble N-ethylmaleimide-sensitive factor-activating protein receptors (SNAREs), etc., are hijacked to evade lysosomal degradation. Legionella, Salmonella, Coxiella, Chlamydia, Mycobacterium, and Brucella are examples of pathogens which diverge from the endocytic pathway by using effector-mediated mechanisms to overcome the challenges and establish their intracellular niches. These pathogens extensively utilise and modulate the end processes of secretory pathways, particularly SNAREs, in repurposing the PCV into specialised compartments resembling the host organelles within the secretory network; at the same time, they avoid being degraded by the host's cellular mechanisms. Here, we discuss the recent research advances on the host-pathogen interaction/crosstalk that involves host SNAREs, conserved cellular processes, and the ongoing host-pathogen defence mechanisms in the molecular arms race against each other. The current knowledge of SNAREs, and intravacuolar bacterial pathogen interactions, enables us to understand host cellular innate immune pathways, maintenance of homeostasis, and potential therapeutic strategies to combat ever-growing antimicrobial resistance.
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Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
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Acanthamoeba castellanii , Legionella pneumophila , Acanthamoeba castellanii/genética , Escherichia coli/genética , Fagocitosis/genética , FagosomasRESUMEN
IMPORTANCE: Candida albicans is a leading human fungal pathogen that often causes life-threatening infections in immunocompromised individuals. The ability of C. albicans to transition between yeast and filamentous forms is key to its virulence, and this occurs in response to many host-relevant cues, including engulfment by host macrophages. While previous efforts identified C. albicans genes required for filamentation in other conditions, the genes important for this morphological transition upon internalization by macrophages remained largely enigmatic. Here, we employed a functional genomic approach to identify genes that enable C. albicans filamentation within macrophages and uncovered a role for the mitochondrial ribosome, respiration, and the SNF1 AMP-activated kinase complex. Additionally, we showed that glucose uptake and glycolysis by macrophages support C. albicans filamentation. This work provides insights into the metabolic dueling that occurs during the interaction of C. albicans with macrophages and identifies vulnerabilities in C. albicans that could serve as promising therapeutic targets.
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[This corrects the article DOI: 10.3389/fcimb.2023.1220089.].
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Phagocytosis is a critical immune function for infection control and tissue homeostasis. During phagocytosis, pathogens are internalized and degraded in phagolysosomes. For pathogens that evade immune degradation, the prevailing view is that virulence factors are required to disrupt the biogenesis of phagolysosomes. In contrast, we present here that physical forces from motile pathogens during cell entry divert them away from the canonical degradative pathway. This altered fate begins with the force-induced remodeling of the phagocytic synapse formation. We used the parasite Toxoplasma gondii as a model because live Toxoplasma actively invades host cells using gliding motility. To differentiate the effects of physical forces from virulence factors in phagocytosis, we employed magnetic forces to induce propulsive entry of inactivated Toxoplasma into macrophages. Experiments and computer simulations show that large propulsive forces hinder productive activation of receptors by preventing their spatial segregation from phosphatases at the phagocytic synapse. Consequently, the inactivated parasites are engulfed into vacuoles that fail to mature into degradative units, similar to the live motile parasite's intracellular pathway. Using yeast cells and opsonized beads, we confirmed that this mechanism is general, not specific to the parasite used. These results reveal new aspects of immune evasion by demonstrating how physical forces during active cell entry, independent of virulence factors, enable pathogens to circumvent phagolysosomal degradation.
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Parásitos , Toxoplasma , Animales , Internalización del Virus , Fagocitosis , Macrófagos , Factores de VirulenciaRESUMEN
Invasive fungal infections pose a major threat to human health. Bacterial and protozoan pathogens secrete protein effectors that overcome innate immune barriers to promote microbial colonization, yet few such molecules have been identified in human fungal pathogens. Recent studies have begun to reveal these long-sought effectors and have illuminated how they subvert key cellular pathways, including apoptosis, myeloid cell polarization, Toll-like receptor signaling, and phagosome action. Thus, despite lacking the specialized secretion systems of bacteria and parasites, it is increasingly clear that fungi independently evolved effectors targeting pathways often subverted by other classes of pathogens. These findings demonstrate the remarkable power of convergent evolution to enable diverse microbes to infect humans while also setting the stage for detailed dissection of fungal disease mechanisms.
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Cystic fibrosis (CF) airway disease is characterized by chronic polymicrobial infections and an infiltration of neutrophils (PMNs). Staphylococcus aureus has been the most prevalent respiratory pathogen in CF. In particular, methicillin-resistant S. aureus (MRSA) represents a huge clinical burden in CF due to its association with lung disease and increased resistance to antibiotics. In CF, PMNs are unable to kill and clear MRSA. The reason for this remains largely unknown. Our study found that CF PMNs are as equally capable of killing MRSA as healthy PMNs. We show that the CF sputum, however, significantly impairs the ability of human PMNs to kill CF MRSA isolates. In the absence of CF sputum, PMNs kill MRSA via intracellular mechanisms mediated by phagocytosis, rather than extracellular mechanisms via NET formation. CF sputum does not affect the phagocytosis of MRSA via healthy or CF PMNs. Our results demonstrate that CF sputum exposure impairs phagosomal levels of reactive oxygen species (ROS) in MRSA-phagocytosing PMNs. While phagosomal co-localizations of MRSA with primary granule markers, myeloperoxidase and cathepsin D, were significantly reduced upon CF sputum exposure, that of a third azurophilic granule marker, neutrophil elastase, remained unaffected. This suggests that CF sputum does not compromise the fusion of primary granules with phagosomes but diminishes phagosomal ROS levels via another, likely more specific, mechanism. Overall, we identified the airway environment as an important factor that restricts neutrophils' oxidative microbicidal activities in CF against MRSA. These results deliver new details of the complex host-pathogen interactions present in the CF lung.
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Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. Human macrophages exposed to Aggregatibacter actinomycetemcomitans (Aa) Cdt respond through canonical and non-canonical inflammasome activation to stimulate cytokine release. The inflammatory response is dependent on PI3K signaling blockade via the toxin's phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity; converting PIP3 to phosphatidylinsoitol-3,4-diphosphate (PI3,4P2) thereby depleting PIP3 pools. Phosphoinositides, also play a critical role in phagosome trafficking, serving as binding domains for effector proteins during phagosome maturation and subsequent fusion with lysosomes. We now demonstrate that AaCdt manipulates the phosphoinositide (PI) pools of phagosome membranes and alters Rab5 association. Exposure of macrophages to AaCdt slowed phagosome maturation and decreased phago-lysosome formation, thereby compromising macrophage phagocytic function. Moreover, macrophages exposed to Cdt showed decreased bactericidal capacity leading to increase in Aggregatibacter actinomycetemcomitans survival. Thus, Cdt may contribute to increased susceptibility to bacterial infection. These studies uncover an underexplored aspect of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt-producing organisms such as Aa.