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Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

ABSTRACT: Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling but is independent of all activity by Toll-like receptors and does not require the presence of the adaptor protein Card9. We further demonstrate that reactive oxygen species generation by NADPH oxidase is required for LC3 recruitment to the fungal phagosome. These observations directly link LC3 to the inflammatory pathway against C. albicans in macrophages.

SUBMITTER: Tam JM

PROVIDER: S-EPMC4271056 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

Tam Jenny M JM Mansour Michael K MK Khan Nida S NS Seward Michael M Puranam Sravanthi S Tanne Antoine A Sokolovska Anna A Becker Christine E CE Acharya Mridu M Baird Michelle A MA Choi Augustine M K AM Davidson Michael W MW Segal Brahm H BH Lacy-Hulbert Adam A Stuart Lynda M LM Xavier Ramnik J RJ Vyas Jatin M JM

The Journal of infectious diseases 20140519 11

Autophagy has been postulated to play role in mammalian host defense against fungal pathogens, although the molecular details remain unclear. Here, we show that primary macrophages deficient in the autophagic factor LC3 demonstrate diminished fungicidal activity but increased cytokine production in response to Candida albicans stimulation. LC3 recruitment to fungal phagosomes requires activation of the fungal pattern receptor dectin-1. LC3 recruitment to the phagosome also requires Syk signaling ...[more]

PMID: 24842831

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Project description:Intracellular pathogens such as Salmonella depend on their molecular virulence factors to evade host defense responses like autophagy. Using a zebrafish systemic infection model, we have previously shown that phagocytes, predominantly macrophages, target Salmonella Typhimurium by an autophagy-related pathway known as Lc3-associated phagocytosis (LAP), which is dependent on the host protein Rubicon. Here, we explore the influence of Salmonella virulence factors on pathogenicity in the zebrafish model and induction of LAP as a defense response. We investigated five mutant strains that all could trigger GFP-Lc3 recruitment as puncta or rings around single bacteria or bacterial clusters, in a Rubicon-dependent manner. We found that S. Typhimurium strains carrying mutations in PhoP or PurA, responsible for adaptation to the intracellular environment and efficient metabolism of purines, respectively, are attenuated in the zebrafish model. However, both strains show increased virulence when LAP is inhibited by knockdown of Rubicon. Mutations in type III secretion systems 1 and 2, SipB and SsrB, which are important for invading and replicating in non-phagocytic cells, did not affect the ability to establish successful infection in the zebrafish model. This observation is in line with our previous characterization of this infection model revealing that macrophages actively phagocytose the majority of S. Typhimurium. In contrast to SipB mutants, SsrB mutants were unable to become more virulent in Rubicon-deficient hosts, suggesting that type III system 2 effectors are important for intracellular replication of Salmonella in the absence of LAP. Finally, we found that mutation of FlhD, required for production of flagella, renders S. Typhimurium hypervirulent both in wild type zebrafish embryos and in Rubicon-deficient hosts. FlhD mutation also led to lower levels of GFP-Lc3 recruitment compared with the wild type strain, indicating that recognition of flagellin by the host innate immune system promotes the LAP response. Together, our results provide new evidence that the Rubicon-dependent LAP process is an important defense mechanism against S. Typhimurium.

Project description:Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Dataset Information

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

Publications

Dectin-1-dependent LC3 recruitment to phagosomes enhances fungicidal activity in macrophages.

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