Imaging invasive fungal growth and inflammation in NADPH oxidase-deficient zebrafish

AbstractNeutrophils are primary cells of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections including those by the generally non-pathogenic fungusAspergillus nidulans. The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response toAspergillus nidulans. In a wild-type host,A. nidulansgerminates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasiveA. nidulansinfection despite robust neutrophil infiltration. Expression of p22phoxspecifically in neutrophils does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasiveA. nidulansinfection.

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FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽

10.1182/blood-2007-11-126029 ◽

2008 ◽

Vol 112 (9) ◽

pp. 3867-3877 ◽

Cited By ~ 63

Author(s):

Wei Tian ◽

Xing Jun Li ◽

Natalie D. Stull ◽

Wenyu Ming ◽

Chang-Il Suh ◽

...

Keyword(s):

Nadph Oxidase ◽

Oxidase Activity ◽

Superoxide Production ◽

Resting Cells ◽

Cellular Activation ◽

Nadph Oxidase Activity ◽

Phagocyte Nadph Oxidase ◽

Before And After ◽

Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

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Sphingolipid biosynthetic pathway is crucial for growth, biofilm formation and membrane integrity of Scedosporium boydii

Future Medicinal Chemistry ◽

10.4155/fmc-2019-0186 ◽

2019 ◽

Vol 11 (22) ◽

pp. 2905-2917 ◽

Cited By ~ 2

Author(s):

Rodrigo Rollin-Pinheiro ◽

Victor Pereira Rochetti ◽

Mariana Ingrid Dutra da Silva Xisto ◽

Livia Cristina Liporagi-Lopes ◽

Beatriz Bastos ◽

...

Keyword(s):

Cell Biology ◽

Pathogenic Fungus ◽

Membrane Integrity ◽

Fungal Growth ◽

Antifungal Drugs ◽

Wide Range ◽

Antifungal Potential ◽

Sphingolipid Biosynthesis ◽

Increased Susceptibility

Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii, a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.

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Structural Differences Influence Biological Properties of Glucosylceramides from Clinical and Environmental Isolates of Scedosporium aurantiacum and Pseudallescheria minutispora

Journal of Fungi ◽

10.3390/jof5030062 ◽

2019 ◽

Vol 5 (3) ◽

pp. 62 ◽

Cited By ~ 1

Author(s):

Adriana Caneppa ◽

Jardel de Meirelles ◽

Rodrigo Rollin-Pinheiro ◽

Mariana Xisto ◽

Livia Liporagi-Lopes ◽

...

Keyword(s):

Fungal Growth ◽

Biological Properties ◽

Ionization Mass ◽

Phagocytic Cells ◽

Pseudallescheria Boydii ◽

Cell Surface Molecule ◽

Host Pathogen Interaction ◽

Host Pathogen ◽

Conserved Epitope

Scedosporium/Lomentospora complex is composed of filamentous fungi, including some clinically relevant species, such as Pseudallescheria boydii, Scedosporium aurantiacum, and Scedosporium apiospermum. Glucosylceramide (GlcCer), a conserved neutral glycosphingolipid, has been described as an important cell surface molecule playing a role in fungal morphological transition and pathogenesis. The present work aimed at the evaluation of GlcCer structures in S. aurantiacum and Pseudallescheria minutispora, a clinical and an environmental isolate, respectively, in order to determine their participation in fungal growth and host-pathogen interactions. Structural analysis by positive ion-mode ESI-MS (electrospray ionization mass spectrometer) revealed the presence of different ceramide moieties in GlcCer in these species. Monoclonal antibodies against Aspergillus fumigatus GlcCer could recognize S. aurantiacum and P. minutispora conidia, suggesting a conserved epitope in fungal GlcCer. In addition, these antibodies reduced fungal viability, enhanced conidia phagocytosis by macrophages, and decreased fungal survival inside phagocytic cells. Purified GlcCer from both species led to macrophage activation, increasing cell viability as well as nitric oxide and superoxide production in different proportions between the two species. These results evidenced some important properties of GlcCer from species of the Scedosporium/Lomentospora complex, as well as the effects of monoclonal anti-GlcCer antibodies on fungal cells and host-pathogen interaction. The differences between the two species regarding the observed biological properties suggest that variation in GlcCer structures and strain origin could interfere in the role of GlcCer in host-pathogen interaction.

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Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group AStreptococcusIs Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin

Infection and Immunity ◽

10.1128/iai.00527-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 2

Author(s):

Benfang Lei ◽

Dylan Minor ◽

Wenchao Feng ◽

Maria Jerome ◽

Mark T. Quinn ◽

...

Keyword(s):

Nadph Oxidase ◽

Skin Infection ◽

Neutrophil Infiltration ◽

Streptococcal Pharyngitis ◽

Nuclear Staining ◽

Wild Type ◽

Phagocyte Nadph Oxidase ◽

Group A ◽

And Control ◽

Dependent Mechanism

ABSTRACTGroup AStreptococcus(GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagAwere full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagAinfection was not cleared. Gp91phoxknockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA−) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

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