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Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages.

Naderer T, Heng J, Saunders EC, Kloehn J, Rupasinghe TW, Brown TJ, McConville MJ - PLoS Pathog. (2015)

Bottom Line: In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages.Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes.These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

View Article: PubMed Central - PubMed

Affiliation: The Department of Biochemistry and Molecular Biology and the Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

No MeSH data available.


Related in: MedlinePlus

Intracellular ∆gnat parasites are rescued by exogenous GlcNAc sources.(A) RAW 264.7 macrophages were infected with wild type (WT) and ∆gnat stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and ∆gnat 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) ∆gnat promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD600.
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ppat.1005136.g005: Intracellular ∆gnat parasites are rescued by exogenous GlcNAc sources.(A) RAW 264.7 macrophages were infected with wild type (WT) and ∆gnat stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and ∆gnat 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) ∆gnat promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD600.

Mentions: To further investigate potential sources of GlcNAc utilized by intracellular amastigotes, infection experiments were undertaken in BALB/c bone marrow-derived macrophages (BMDMs). ∆gnat promastigotes were rapidly cleared by BMDMs within four days post infection. In contrast, wild type parasite levels remained constant over the same period (Fig 4D). Similar to promastigotes, lesion derived ∆gnat amastigotes were unable to survive in cultured macrophages (Fig 4E), suggesting that ex vivo macrophages, but not macrophages in skin lesions, fail to provide sufficient levels of GlcNAc to support intracellular ∆gnat growth. To investigate whether intracellular survival of ∆gnat parasites could be rescued by exogenous GlcNAc, the medium of infected macrophages was supplemented with 50 μg/ml or 500 μg/ml GlcNAc. Addition of physiologically relevant concentrations of GlcNAc (~40 μg/ml in serum [15]) did not rescue growth of the ∆gnat mutant, while addition of a 10-fold higher concentration GlcNAc resulted in a modest increase (2-fold) in intracellular growth (Fig 5A). These results suggest that the fluid phase uptake of exogenous GlcNAc and/or transport of GlcNAc from the macrophage cytoplasm to the phagolysosome contribute minimally to the observed ∆gnat growth in skin lesions.


Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages.

Naderer T, Heng J, Saunders EC, Kloehn J, Rupasinghe TW, Brown TJ, McConville MJ - PLoS Pathog. (2015)

Intracellular ∆gnat parasites are rescued by exogenous GlcNAc sources.(A) RAW 264.7 macrophages were infected with wild type (WT) and ∆gnat stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and ∆gnat 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) ∆gnat promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD600.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559419&req=5

ppat.1005136.g005: Intracellular ∆gnat parasites are rescued by exogenous GlcNAc sources.(A) RAW 264.7 macrophages were infected with wild type (WT) and ∆gnat stationary promastigotes in the presence of either GlcNAc, hyaluronic acid (HA) or chitin. Intracellular growth was determined after fixation and staining with the DNA dye, Hoechst, and is expressed relative to parasite numbers at day four of untreated cells (whereby WT contained 67 +/- 8 parasites and ∆gnat 26 +/- 4 parasites). Error bars are the SD from three biological repeat experiments and p-values were calculated by the Student’s t-test. (B) WT and (C) ∆gnat promastigotes were cultured in the presence of GlcNAc, HA or chitin as the sole hexose source and growth was determined by OD600.
Mentions: To further investigate potential sources of GlcNAc utilized by intracellular amastigotes, infection experiments were undertaken in BALB/c bone marrow-derived macrophages (BMDMs). ∆gnat promastigotes were rapidly cleared by BMDMs within four days post infection. In contrast, wild type parasite levels remained constant over the same period (Fig 4D). Similar to promastigotes, lesion derived ∆gnat amastigotes were unable to survive in cultured macrophages (Fig 4E), suggesting that ex vivo macrophages, but not macrophages in skin lesions, fail to provide sufficient levels of GlcNAc to support intracellular ∆gnat growth. To investigate whether intracellular survival of ∆gnat parasites could be rescued by exogenous GlcNAc, the medium of infected macrophages was supplemented with 50 μg/ml or 500 μg/ml GlcNAc. Addition of physiologically relevant concentrations of GlcNAc (~40 μg/ml in serum [15]) did not rescue growth of the ∆gnat mutant, while addition of a 10-fold higher concentration GlcNAc resulted in a modest increase (2-fold) in intracellular growth (Fig 5A). These results suggest that the fluid phase uptake of exogenous GlcNAc and/or transport of GlcNAc from the macrophage cytoplasm to the phagolysosome contribute minimally to the observed ∆gnat growth in skin lesions.

Bottom Line: In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages.Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes.These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

View Article: PubMed Central - PubMed

Affiliation: The Department of Biochemistry and Molecular Biology and the Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, Parkville, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites.

No MeSH data available.


Related in: MedlinePlus