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      Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH

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          Abstract

          Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

          Author summary

          Cryptococcus neoformans is a relatively frequent cause of life-threatening infection in severely immunocompromised patients, especially those with AIDS. Persistence of infection involves residence within macrophages, where C. neoformans can survive and replicate while residing in the phagolysosome. New treatments may be developed from a better understanding of how this pathogen resists clearance from and adapts for persistence in host phagocytic cells. In this study, we demonstrate a novel role for urease, a major virulence factor of C. neoformans, in its interaction with macrophages. This enzyme is able to break down urea into ammonia, which is a base, thus raising the surrounding pH. In the context of a mammalian infection, we show that cryptococcal urease increases the phagolysosomal pH which delays yeast replication, therefore causing less damage to macrophages and prolongs intracellular residence. Moreover, urease promotes C. neoformans exit from macrophages without killing the host cells. Overall, our data implies that urease also contributes to virulence by allowing the pathogen to persist and disseminate in macrophages.

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          Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages.

          Cryptococcus neoformans (Cn) is a soil fungus that causes life-threatening meningitis in immunocompromised patients and is a facultative intracellular pathogen capable of replication inside macrophages. The mechanism by which environmental fungi acquire and maintain virulence for mammalian hosts is unknown. We hypothesized that the survival strategies for Cn after ingestion by macrophages and amoebae were similar. Microscopy, fungal and amoebae killing assays, and phagocytosis assays revealed that Cn is phagocytosed by and replicates in Acanthamoeba castellanii, which leads to death of amoebae. An acapsular strain of Cn did not survive when incubated with amoebae, but melanization protected these cells against killing by amoebae. A phospholipase mutant had a decreased replication rate in amoebae compared with isogenic strains. These observations suggest that cryptococcal characteristics that contribute to mammalian virulence also promote fungal survival in amoebae. Intracellular replication was accompanied by the accumulation of polysaccharide containing vesicles similar to those described in Cn-infected macrophages. The results suggest that the virulence of Cn for mammalian cells is a consequence of adaptations that have evolved for protection against environmental predators such as amoebae and provide an explanation for the broad host range of this pathogenic fungus.
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            Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.

            Cryptococcus neoformans (Cn) is an encapsulated yeast that is a facultative intracellular pathogen and a frequent cause of human disease. The interaction of Cn with alveolar macrophages is critical for containing the infection , but Cn can also replicate intracellularly and lyse macrophages . Cn has a unique intracellular pathogenic strategy that involves cytoplasmic accumulation of polysaccharide-containing vesicles and intracellular replication leading to the formation of spacious phagosomes in which multiple cryptococcal cells are present . The Cn intracellular pathogenic strategy in macrophages and amoebas is similar, leading to the proposal that it originated as a mechanism for survival against phagocytic predators in the environment . Here, we report that under certain conditions, including phagosomal maturation, possible actin depolymerization, and homotypic phagosome fusion, Cn can exit the macrophage host through an extrusion of the phagosome, while both the released pathogen and host remain alive and able to propagate. The phenomenon of "phagosomal extrusion" indicates the existence of a previously unrecognized mechanism whereby a fungal pathogen can escape the intracellular confines of mammalian macrophages to continue propagation and, possibly, dissemination.
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              Evidence of a role for monocytes in dissemination and brain invasion by Cryptococcus neoformans.

              The pathogenesis of cryptococcosis, including the events leading to the production of meningoencephalitis, is still largely unknown. Evidence of a transcellular passage of Cryptococcus neoformans across the blood-brain barrier (BBB) and subsequent BBB disruption exists, but the paracellular passage of free yeasts and the role of monocytes in yeast dissemination and brain invasion (Trojan horse method) remain uncertain. We used our model of disseminated cryptococcosis, in which crossing of the BBB starts 6 h after intravenous inoculation, to study paracellular passage of the BBB. We prepared bone marrow-derived monocytes (BMDM) infected in vitro with C. neoformans (BMDM yeasts) and free yeasts and measured fungal loads in tissues. (i) Spleen and lung CFU were >2-fold higher in mice treated with BMDM yeasts than in those treated with free yeasts for 1 and 24 h (P < 0.05), while brain CFU were increased (3.9 times) only at 24 h (P < 0.05). (ii) By comparing the kinetics of brain invasion in naïve mice and in mice with preestablished cryptococcosis, we found that CFU were lower in the latter case, except at 6 h, when CFU from mice inoculated with BMDM yeasts were comparable to those measured in naïve mice and 2.5-fold higher than those in mice with preestablished cryptococcosis who were inoculated with free yeasts. (iii) Late phagocyte depletion obtained by clodronate injection reduced disease severity and lowered the fungal burden by 40% in all organs studied. These results provide evidence for Trojan horse crossing of the BBB by C. neoformans, together with mechanisms involving free yeasts, and overall for a role of phagocytes in fungal dissemination.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: Methodology
                Role: InvestigationRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                15 June 2018
                June 2018
                : 14
                : 6
                Affiliations
                [1 ] Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America
                [2 ] Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America
                University of Birmingham, UNITED KINGDOM
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PPATHOGENS-D-18-00560
                10.1371/journal.ppat.1007144
                6021110
                29906292
                © 2018 Fu et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 9, Tables: 0, Pages: 31
                Product
                Funding
                This study was supported by National Institutes of Health (AC) grants R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. ( https://grants.nih.gov/funding/index.htm) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Ureases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Ureases
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Cryptococcus
                Cryptococcus Neoformans
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Fungal Pathogens
                Cryptococcus Neoformans
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Fungal Pathogens
                Cryptococcus Neoformans
                Biology and Life Sciences
                Mycology
                Fungal Pathogens
                Cryptococcus Neoformans
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Urea
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Urea
                Biology and Life Sciences
                Organisms
                Eukaryota
                Fungi
                Cryptococcus
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Exocytosis
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Exocytosis
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Phagocytosis
                Medicine and Health Sciences
                Infectious Diseases
                Fungal Diseases
                Yeast Infections
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-06-27
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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