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Nature Jun 2024Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms. During phagocytosis, innate immune receptors and...
Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.
Topics: Phagosomes; B7-H1 Antigen; Animals; Mice; Macrophages; Ribosomal Proteins; Protein Binding; Interleukin-10; Phagocytosis; Saccharomyces cerevisiae; Fungal Proteins; Ligands; Humans; Female; Immunity, Innate
PubMed: 38839956
DOI: 10.1038/s41586-024-07499-6 -
Science Advances Jun 2024Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic...
Tissue stiffening is a predominant feature of fibrotic disorders, but the response of macrophages to changes in tissue stiffness and cellular context in fibrotic diseases remains unclear. Here, we found that the mechanosensitive ion channel Piezo1 was up-regulated in hepatic fibrosis. Macrophages lacking Piezo1 showed sustained inflammation and impaired spontaneous resolution of early liver fibrosis. Further analysis revealed an impairment of clearance of apoptotic cells by macrophages in the fibrotic liver. Macrophages showed enhanced efferocytosis when cultured on rigid substrates but not soft ones, suggesting stiffness-dependent efferocytosis of macrophages required Piezo1 activation. Besides, Piezo1 was involved in the efficient acidification of the engulfed cargo in the phagolysosomes and affected the subsequent expression of anti-inflammation genes after efferocytosis. Pharmacological activation of Piezo1 increased the efferocytosis capacity of macrophages and accelerated the resolution of inflammation and fibrosis. Our study supports the antifibrotic role of Piezo1-mediated mechanical sensation in liver fibrosis, suggesting that targeting PIEZO1 to enhance macrophage efferocytosis could induce fibrosis regression.
Topics: Ion Channels; Liver Cirrhosis; Animals; Macrophages; Phagocytosis; Mice; Humans; Apoptosis; Mice, Inbred C57BL; Disease Models, Animal; Efferocytosis
PubMed: 38838160
DOI: 10.1126/sciadv.adj3289 -
Cellular and Molecular Life Sciences :... Jun 2024Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular...
Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.
Topics: Ubiquitination; Legionella pneumophila; Humans; Phagosomes; SNARE Proteins; rab GTP-Binding Proteins; Bacterial Proteins; Animals; Qa-SNARE Proteins; Ubiquitin-Protein Ligases; Vacuoles; HEK293 Cells; Mice; rab7 GTP-Binding Proteins; Monomeric GTP-Binding Proteins; Endoplasmic Reticulum
PubMed: 38836877
DOI: 10.1007/s00018-024-05271-7 -
ELife Jun 2024During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures....
During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
Topics: Qa-SNARE Proteins; Autophagosomes; Phosphatidylinositol Phosphates; Humans; Molecular Dynamics Simulation; Autophagy
PubMed: 38831696
DOI: 10.7554/eLife.92189 -
ELife Jun 2024A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during...
A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.
Topics: Autophagy; Autophagosomes; Membrane Fusion; SNARE Proteins; Humans; Animals
PubMed: 38831693
DOI: 10.7554/eLife.99181 -
BioRxiv : the Preprint Server For... May 2024The complex contains opportunistic pathogens that cause chronic infections and inflammation in lungs of people with cystic fibrosis. Two closely related species within...
The complex contains opportunistic pathogens that cause chronic infections and inflammation in lungs of people with cystic fibrosis. Two closely related species within this complex are and the recently classified and encode a type VI secretion system and the effector TecA, which is detected by the pyrin/caspase-1 inflammasome, and triggers macrophage inflammatory death. In our earlier study the pyrin inflammasome was dispensable for lung inflammation in mice infected with AU1054, indicating this species activates an alternative pathway of macrophage inflammatory death. Notably, J2315 and K56-2 can damage macrophage phagosomes and K56-2 triggers activation of the caspase-11 inflammasome, which detects cytosolic LPS. Here we investigated inflammatory cell death in pyrin-deficient ( ) mouse macrophages infected with J2315 or K56-2 or AU1054 or PC184. Macrophage inflammatory death was measured by cleavage of gasdermin D protein, release of cytokines IL-1α and IL-1β and plasma membrane rupture. Findings suggest that J2315 and K56-2 are detected by the caspase-11 inflammasome in macrophages, resulting in IL-1β release. In contrast, inflammasome activation is not detected in macrophages infected with AU1054 or PC184. Instead, AU1054 triggers an alternative macrophage inflammatory death pathway that requires TecA and results in plasma membrane rupture and IL-1α release. Amino acid variation between TecA isoforms in and may explain how the latter species triggers a non-inflammasome macrophage death pathway.
PubMed: 38826213
DOI: 10.1101/2023.09.28.559184 -
Frontiers in Cellular and Infection... 2024Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals...
Parasites possess remarkable abilities to evade and manipulate the immune response of their hosts. is a parasitic tapeworm that causes cystic echinococcosis in animals and humans. The hydatid fluid released by the parasite is known to contain various immunomodulatory components that manipulate host´s defense mechanism. In this study, we focused on understanding the effect of hydatid fluid on dendritic cells and its impact on autophagy induction and subsequent T cell responses. Initially, we observed a marked downregulation of two C-type lectin receptors in the cell membrane, CLEC9A and CD205 and an increase in lysosomal activity, suggesting an active cellular response to hydatid fluid. Subsequently, we visualized ultrastructural changes in stimulated dendritic cells, revealing the presence of macroautophagy, characterized by the formation of autophagosomes, phagophores, and phagolysosomes in the cell cytoplasm. To further elucidate the underlying molecular mechanisms involved in hydatid fluid-induced autophagy, we analyzed the expression of autophagy-related genes in stimulated dendritic cells. Our results demonstrated a significant upregulation of and , indicating the induction of autophagy machinery in response to hydatid fluid exposure. Additionally, using confocal microscopy, we observed an accumulation of LC3 in dendritic cell autophagosomes, confirming the activation of this catabolic pathway associated with antigen presentation. Finally, to evaluate the functional consequences of hydatid fluid-induced autophagy in DCs, we evaluated cytokine transcription in the splenocytes. Remarkably, a robust polyfunctional T cell response, with inhibition of Th2 profile, is characterized by an increase in the expression of and genes. These findings suggest that hydatid fluid-induced autophagy in dendritic cells plays a crucial role in shaping the subsequent T cell responses, which is important for a better understanding of host-parasite interactions in cystic echinococcosis.
Topics: Dendritic Cells; Animals; Echinococcus granulosus; Autophagy; Echinococcosis; T-Lymphocytes; Mice; Lectins, C-Type; Cytokines; Female; Autophagosomes
PubMed: 38817444
DOI: 10.3389/fcimb.2024.1334211 -
MSphere Jun 2024is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system,...
is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved wild-type and mutant Δ or Δ strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the mutation L424I preserves Pma1 H-ATPase activity under high Cu concentrations and that the mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of adaptation to diverse environmental challenges.IMPORTANCE is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.
Topics: Aspergillus fumigatus; Copper; Animals; Mice; Fungal Proteins; Aspergillosis; Mutation; Drug Resistance, Fungal; Virulence; Evolution, Molecular; Glutamate-Cysteine Ligase; Female; Proton-Translocating ATPases
PubMed: 38814077
DOI: 10.1128/msphere.00253-24 -
ELife May 2024(Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the...
(Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6's mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH-dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.
Topics: Humans; Antigens, Bacterial; Bacterial Proteins; Hydrogen-Ion Concentration; Macrophages; Molecular Dynamics Simulation; Mycobacterium tuberculosis; Phagosomes; Single-Domain Antibodies
PubMed: 38805257
DOI: 10.7554/eLife.91930 -
Frontiers in Microbiology 2024() genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis...
() genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using comparative genome analysis, we identified one of the genes, Rv1509, as a signature protein exclusively present in . To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in () constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, and studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in pathogenesis.
PubMed: 38803374
DOI: 10.3389/fmicb.2024.1344857