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Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.

ABSTRACT: In previous studies we showed that a Cryptococcus neoformans mutant lacking glucosylceramide (Deltagcs1) is avirulent and unable to reach the brain when it is administered intranasally into an immunocompetent mouse and is contained in a lung granuloma. To determine whether granuloma formation is key for containment of C. neoformans Deltagcs1, we studied the role of C. neoformans glucosylceramide in a T- and NK-cell-immunodeficient mouse model (Tgepsilon26) in which alveolar macrophages (AMs) are not activated and granuloma formation is not expected. The results show that Tgepsilon26 mice infected with Deltagcs1 do not produce a lung granuloma and that the Deltagcs1 mutant proliferates in the lungs and does disseminate to the brain, although its virulence phenotype is dramatically reduced. Since Deltagcs1 can grow only in acidic niches, such as the phagolysosome of AMs, and not in neutral or alkaline environments, such as the extracellular spaces, we hypothesize that in immunodeficient mice Deltagcs1 proliferates inside AMs. Indeed, we found that depletion of AMs significantly improved Tgepsilon26 mouse survival and decreased the dissemination of Deltagcs1 cells to the central nervous system. Thus, these results suggest that the growth of Deltagcs1 in immunodeficient mice is maintained within AMs. This study highlights the hypothesis that AMs may exacerbate C. neoformans infection in conditions in which there is severe host immunodeficiency.

SUBMITTER: Kechichian TB

PROVIDER: S-EPMC2044542 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.

Kechichian Talar B TB Shea John J Del Poeta Maurizio M

Infection and immunity 20070730 10

In previous studies we showed that a Cryptococcus neoformans mutant lacking glucosylceramide (Deltagcs1) is avirulent and unable to reach the brain when it is administered intranasally into an immunocompetent mouse and is contained in a lung granuloma. To determine whether granuloma formation is key for containment of C. neoformans Deltagcs1, we studied the role of C. neoformans glucosylceramide in a T- and NK-cell-immunodeficient mouse model (Tgepsilon26) in which alveolar macrophages (AMs) are ...[more]

PMID: 17664261

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Project description:The sphingolipid glucosylceramide (GlcCer) and factors involved in the fungal GlcCer pathways were shown earlier to be an integral part of fungal virulence, especially in fungal replication at 37 °C, in neutral/alkaline pH and 5 % CO2 environments (e.g. alveolar spaces). Two mutants, ?gcs 1 lacking glucosylceramide synthase 1 gene (GCS1) which catalyzes the formation of sphingolipid GlcCer from the C9-methyl ceramide and ?smt1 lacking sphingolipid C9 methyltransferase gene (SMT1), which adds a methyl group to position nine of the sphingosine backbone of ceramide, of this pathway were attenuated in virulence and have a growth defect at the above-mentioned conditions. These mutants with either no or structurally modified GlcCer located on the cell-membrane have reduced membrane rigidity, which may have altered not only the physical location of membrane proteins but also their expression, as the pathogen's mode of adaptation to changing need. Importantly, pathogens are known to adapt themselves to the changing host environments by altering their patterns of gene expression.By transcriptional analysis of gene expression, we identified six genes whose expression was changed from their wild-type counterpart grown in the same conditions, i.e. they became either down regulated or up regulated in these two mutants. The microarray data was validated by real-time PCR, which confirmed their fold change in gene expression. All the six genes we identified, viz siderochrome-iron transporter (CNAG_02083), monosaccharide transporter (CNAG_05340), glucose transporter (CNAG_03772), membrane protein (CNAG_03912), membrane transport protein (CNAG_00539), and sugar transporter (CNAG_06963), are membrane-localized and have significantly altered gene expression levels. Therefore, we hypothesize that these genes function either independently or in tandem with a structurally modified cell wall/plasma membrane resulting from the modifications of the GlcCer pathway and thus possibly disrupt transmembrane signaling complex, which in turn contributes to cryptococcal osmotic, pH, ion homeostasis and its pathobiology.Six genes identified from gene expression microarrays by gene set enrichment analysis and validated by RT-PCR, are membrane located and associated with the growth defect at neutral-alkaline pH due to the absence and or presence of a structurally modified GlcCer. They may be involved in the transmembrane signaling network in Cryptococcus neoformans, and therefore the pathobiology of the fungus in these conditions.

Project description:Cryptococcus-macrophage interaction is crucial in the development of cryptococcocal diseases. C. neoformans and C. gattii are major pathogenic species that occupy different niches and cause different clinical manifestations. However, the differences of macrophage interaction among these species in affecting different disease outcomes and immune responses have not been clearly addressed. Here, we examined the macrophage uptake rates, intracellular loads and intracellular proliferation rates of C. neoformans and C. gattii clinical isolates from Thailand and analyzed the effect of those interactions on fungal burdens and host immune responses. C. neoformans isolates showed a higher phagocytosis rate but lower intracellular proliferation rate than C. gattii. Indeed, the high intracellular proliferation rate of C. gattii isolates did not influence the fungal burdens in lungs and brains of infected mice, whereas infection with high-uptake C. neoformans isolates resulted in significantly higher brain burdens that associated with reduced survival rate. Interestingly, alveolar macrophages of mice infected with high-uptake C. neoformans isolates showed distinct patterns of alternatively activated macrophage (M2) gene expressions with higher Arg1, Fizz1, Il13 and lower Nos2, Ifng, Il6, Tnfa, Mcp1, csf2 and Ip10 transcripts. Corresponding to this finding, infection with high-uptake C. neoformans resulted in enhanced arginase enzyme activity, elevated IL-4 and IL-13 and lowered IL-17 in the bronchoalveolar lavage. Thus, our data suggest that the macrophage interaction with C. neoformans and C. gattii may affect different disease outcomes and the high phagocytosis rates of C. neoformans influence the induction of type-2 immune responses that support fungal dissemination and disease progression. Abbreviation: Arg1: Arginase 1; BAL: Bronchoalveolar lavage; CCL17: Chemokine (C-C motif) ligand 17; CNS: Central nervous system; CSF: Cerebrospinal fluid; Csf2: Colony-stimulating factor 2; Fizz1: Found in inflammatory zone 1; HIV: Human immunodeficiency virus; ICL: Intracellular cryptococcal load; Ifng: Interferon gamma; Ip10: IFN-g-inducible protein 10; IPR: Intracellular proliferation rate; Mcp1: Monocyte chemoattractant protein 1; Nos2: Nitric oxide synthase 2; PBS: Phosphate-Buffered Saline; Th: T helper cell; Tnfa: Tumor necrosis factor alpha.

Dataset Information

Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.

Publications

Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice.

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