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  • 1. Achouiti, A.
    et al.
    Vogl, T.
    Urban, Constantin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hommes, T. J.
    van Zoelen, M. A.
    Florquin, S.
    Roth, J.
    van 't Veer, C.
    de Vos, A. F.
    van der Poll, T.
    Myeloid related protein (mrp) 8/14 contributes to an antibacterial host response against klebsiella (k.) pneumoniae2012In: Shock, ISSN 1073-2322, E-ISSN 1540-0514, Vol. 37, no S1, p. 56-56Article in journal (Other academic)
  • 2. Achouiti, Ahmed
    et al.
    Vogl, Thomas
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Röhm, Marc
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Hommes, Tijmen J
    van Zoelen, Marieke AD
    Florquin, Sandrine
    Roth, Johannes
    van't Veer, Cornelis
    de Vos, Alex F
    van der Poll, Tom
    Myeloid-related protein-14 contributes to protective immunity in gram-negative pneumonia derived sepsis2012In: PLoS Pathogens, ISSN 1553-7374, Vol. 8, no 10, p. e1002987-Article in journal (Refereed)
    Abstract [en]

    Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14(-/-)) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14(-/-) macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14(-/-) neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.

  • 3.
    Almyroudis, Nikolaos G.
    et al.
    Roswell Park Cancer Institute, Buffalo, New York, United States of America.
    Grimm, Melissa J.
    Roswell Park Cancer Institute, Buffalo, New York, United States of America.
    Davidson, Bruce A.
    Roswell Park Cancer Institute, Buffalo, New York, United States of America.
    Röhm, Marc
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Segal, Brahm H.
    Roswell Park Cancer Institute, Buffalo, New York, United States of America.
    NETosis and NADPH oxidase: at the intersection of host defense, inflammation, and injury2013In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 4, article id 45Article, review/survey (Refereed)
    Abstract [en]

    Neutrophils are armed with both oxidant-dependent and -independent pathways for killing pathogens. Activation of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase constitutes an emergency response to infectious threat and results in the generation of antimicrobial reactive oxidants. In addition, NADPH oxidase activation in neutrophils is linked to activation of granular proteases and generation of neutrophil extracellular traps (NETs). NETosis involves the release of nuclear and granular components that can target extracellular pathogens. NETosis is activated during microbial threat and in certain conditions mimicking sepsis, and can result in both augmented host defense and inflammatory injury. In contrast, apoptosis, the physiological form of neutrophil death, not only leads to non-inflammatory cell death but also contributes to alleviate inflammation. Although there are significant gaps in knowledge regarding the specific contribution of NETs to host defense, we speculate that the coordinated activation of NADPH oxidase and NETosis maximizes microbial killing. Work in engineered mice and limited patient experience point to varying susceptibility of bacterial and fungal pathogens to NADPH oxidase versus NET constituents. Since reactive oxidants and NET constituents can injure host tissue, it is important that these pathways be tightly regulated. Recent work supports a role for NETosis in both acute lung injury and in autoimmunity. Knowledge gained about mechanisms that modulate NETosis may lead to novel therapeutic approaches to limit inflammation-associated injury.

  • 4.
    Bianchi, Matteo
    et al.
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Niemiec, Maria J.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Siler, Ulrich
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Reichenbach, Janine
    Division of Immunology/Hematology/BMT, University Children’s Hospital, Zurich, Switzerland .
    Redundant ability of phagocytes to kill Aspergillus species: Reply2011In: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 128, no 3, p. 687-688Article in journal (Refereed)
  • 5. Bianchi, Matteo
    et al.
    Niemiec, Maria J
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Siler, Ulrich
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Reichenbach, Janine
    Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent2011In: Journal of Allergy and Clinical Immunology, ISSN 0091-6749, E-ISSN 1097-6825, Vol. 127, no 5, p. 1243-1252 e.7Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function.

    OBJECTIVE: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD.

    METHODS: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or alpha-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils.

    RESULTS: We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice.

    CONCLUSION: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.

  • 6. Bjornsdottir, Halla
    et al.
    Christenson, Karin
    Forsman, Huamei
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Urban, Constantin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Dahlgren, Claes
    Karlsson, Anna
    Bylund, Johan
    Cytotoxic Peptides from S. aureus Cause Neutrophil Cell Death with NET-like Features2014In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 79, no 6, p. 432-432Article in journal (Other academic)
  • 7. Bjornsdottir, Halla
    et al.
    Rudin, Agnes Dahlstrand
    Klose, Felix P.
    Elmwall, Jonas
    Welin, Amanda
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Christenson, Karin
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Forsman, Huamei
    Dahlgren, Claes
    Karlsson, Anna
    Bylund, Johan
    Phenol-soluble Modulin α Peptide Toxins from aggressive Staphylococcus aureus induce rapid Formation of neutrophil extracellular Traps through a reactive Oxygen species-independent Pathway2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 257Article in journal (Refereed)
    Abstract [en]

    Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin alpha (PSM alpha) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMa-induced NETs contained the typical protein markers and were able to capture microbes. The PSMa-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMa-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4 degrees C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMa peptides, a process that may be of importance for CA-MRSA virulence.

  • 8. Crawford, Aaron C.
    et al.
    Lehtovirta-Morley, Laura E.
    Alamir, Omran
    Niemiec, Maria Joanna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology–Hans Knoell Institute, Jena, Germany; Center for Sepsis Control and Care (CSCC), University Hospital, Jena, Germany.
    Alawfi, Bader
    Alsarraf, Mohammad
    Skrahina, Volha
    Costa, Anna C. B. P.
    Anderson, Andrew
    Yellagunda, Sujan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ballou, Elizabeth R.
    Hube, Bernhard
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wilson, Duncan
    Biphasic zinc compartmentalisation in a human fungal pathogen2018In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 14, no 5, article id e1007013Article in journal (Refereed)
    Abstract [en]

    Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2 Delta growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrtl/Zrt2-cellular import, followed by Zrcl-dependent intracellular compartmentalisation.

  • 9. De Samber, Bjorn
    et al.
    Niemiec, Maria J.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Microbial Immunology Research Group, Hans Knöll Institute / Leibniz-Institute for Natural Product Research and Infection Biology, Jena, Germany.
    Laforce, Brecht
    Garrevoet, Jan
    Vergucht, Eva
    De Rycke, Riet
    Cloetens, Peter
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vincze, Laszlo
    Probing Intracellular Element Concentration Changes during Neutrophil Extracellular Trap Formation Using Synchrotron Radiation Based X-Ray Fluorescence2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 11, article id e0165604Article in journal (Refereed)
    Abstract [en]

    High pressure frozen (HPF), cryo-substituted microtome sections of 2 mu m thickness containing human neutrophils (white blood cells) were analyzed using synchrotron radiation based X-ray fluorescence (SR nano-XRF) at a spatial resolution of 50 nm. Besides neutrophils from a control culture, we also analyzed neutrophils stimulated for 1-2 h with phorbol myristate acetate (PMA), a substance inducing the formation of so-called Neutrophil Extracellular Traps (or NETs), a defense system again pathogens possibly involving proteins with metal chelating properties. In order to gain insight in metal transport during this process, precise local evaluation of elemental content was performed reaching limits of detection (LODs) of 1 ppb. Mean weight fractions within entire neutrophils, their nuclei and cytoplasms were determined for the three main elements P, S and Cl, but also for the 12 following trace elements: K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Sr and Pb. Statistical analysis, including linear regression provided objective analysis and a measure for concentration changes. The nearly linear Ca and Cl concentration changes in neutrophils could be explained by already known phenomena such as the induction of Ca channels and the uptake of Cl under activation of NET forming neutrophils. Linear concentration changes were also found for P, S, K, Mn, Fe, Co and Se. The observed linear concentration increase for Mn could be related to scavenging of this metal from the pathogen by means of the neutrophil protein calprotectin, whereas the concentration increase of Se may be related to its antioxidant function protecting neutrophils from the reactive oxygen species they produce against pathogens. We emphasize synchrotron radiation based nanoscopic X-ray fluorescence as an enabling analytical technique to study changing (trace) element concentrations throughout cellular processes, provided accurate sample preparation and data-analysis.

  • 10.
    Ermert, David
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Niemiec, Maria Joanna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Röhm, Marc
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Glenthøj, Andreas
    Department of Hematology, National University Hospital, Copenhagen, Denmark..
    Borregaard, Niels
    Department of Hematology, National University Hospital, Copenhagen, Denmark..
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Candida albicans escapes from mouse neutrophils2013In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 94, no 2, p. 223-236Article in journal (Refereed)
    Abstract [en]

    Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

  • 11.
    Ermert, David
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Laube, Britta
    Goosmann, Christian
    Zychlinsky, Arturo
    Brinkmann, Volker
    Mouse neutrophil extracellular traps in microbial infections.2009In: Journal of innate immunity, ISSN 1662-8128, Vol. 1, no 3, p. 181-193Article in journal (Refereed)
    Abstract [en]

    Neutrophil extracellular traps (NETs) play an important role in innate immunity to microbial infections. NETs have been described in several species, but the molecular details of NET formation and their role in infection has not been addressed, partly because we lack optimal experimental models. Here we describe tools to investigate NET formation in neutrophils isolated from mice. Upon in vitro stimulation of wild-type mouse neutrophils with PMA, we analyzed 3 important steps in the process of NET formation: reactive oxygen species (ROS) production, NET cell death and NET release. As expected, neutrophils from NADPH oxidase-deficient mice failed to produce ROS and did not die nor release NETs upon stimulation. We found that neutrophils from several mouse strains produced NETs with different efficiency and that NET formation correlated with the amount of ROS produced. Activation with Candida albicans also resulted in ROS production and NET cell death. The hyphal form of this fungus induced NETs more effectively than the yeast form. With this work, we provide tools to study in vitro NET assembly in the mouse system.

  • 12. Ermert, David
    et al.
    Zychlinsky, Arturo
    Urban, Constantin
    Fungal and bacterial killing by neutrophils2009In: Host-Pathogen Interactions: Methods and Protocols / [ed] Steffen Rupp, Kai Sohn, Humana Press, 2009, Vol. 470, p. 293-312Chapter in book (Refereed)
    Abstract [en]

    Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.

  • 13.
    Gillenius, Erik
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The adhesive protein invasin of Yersinia pseudotuberculosis induces neutrophil extracellular traps via β1 integrins2015In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 17, no 5, p. 327-336Article in journal (Refereed)
    Abstract [en]

    Yersinia pseudotuberculosis adhesive protein invasin is crucial for the bacteria to cross the intestine epithelium by binding to β1 integrins on M-cells and gaining access to the underlying tissues. After the crossing invasin can bind to β1 integrins on other cell surfaces, however effector proteins delivered by the type III secretion system Y. pseudotuberculosis efficiently inhibit potential immune responses induced by this interaction. Here, we use mutant Y. pseudotuberculosis strains lacking the type III secretion system and additionally invasin-expressing Escherichia coli to analyze neutrophil responses towards invasin. Our data reveals that invasin induces production of reactive oxygen species and release of chromatin into the extracellular milieu, which we confirmed to be neutrophil extracellular traps by immunofluorescence microscopy. This was mediated through β1 integrins and was dependent on both the production of reactive oxygen species and signaling through phosphoinositide 3-kinase. We therefore have gained insight into a potential role of integrins in inflammation and infection clearance that has not previously been described, suggesting that targeting of β1 integrins could be utilized as an adjunctive therapy against yersiniosis.

  • 14. Ginley, Brandon
    et al.
    Emmons, Tiffany
    Sasankan, Prabhu
    Urban, Constantin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Segal, Brahm H.
    Sarder, Pinaki
    Identification and characterization of neutrophil extracellular trap shapes in flow cytometry2017In: Medical Imaging 2017: Digital Pathology / [ed] Gurcan, MN Tomaszewski, JE, 2017, article id 101400DConference paper (Refereed)
    Abstract [en]

    Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.

  • 15. Ginley, Brandon G
    et al.
    Emmons, Tiffany
    Lutnick, Brendon
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Segal, Brahm H
    Sarder, Pinaki
    Computational detection and quantification of human and mouse neutrophil extracellular traps in flow cytometry and confocal microscopy2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 17755Article in journal (Refereed)
    Abstract [en]

    Neutrophil extracellular traps (NETs) are extracellular defense mechanisms used by neutrophils, where chromatin is expelled together with histones and granular/cytoplasmic proteins. They have become an immunology hotspot, implicated in infections, but also in a diverse array of diseases such as systemic lupus erythematosus, diabetes, and cancer. However, the precise assessment of in vivo relevance in different disease settings has been hampered by limited tools to quantify occurrence of extracellular traps in experimental models and human samples. To expedite progress towards improved quantitative tools, we have developed computational pipelines to identify extracellular traps from an in vitro human samples visualized using the ImageStream® platform (Millipore Sigma, Darmstadt, Germany), and confocal images of an in vivo mouse disease model of aspergillus fumigatus pneumonia. Our two in vitro methods, tested on n = 363/n =145 images respectively, achieved holdout sensitivity/specificity 0.98/0.93 and 1/0.92. Our unsupervised method for thin lung tissue sections in murine fungal pneumonia achieved sensitivity/specificity 0.99/0.98 in n = 14 images. Our supervised method for thin lung tissue classified NETs with sensitivity/specificity 0.86/0.90. We expect that our approach will be of value for researchers, and have application in infectious and inflammatory diseases.

  • 16. Grimm, Melissa J
    et al.
    Vethanayagam, R Robert
    Almyroudis, Nikolaos G
    Dennis, Carly G
    Khan, A Nazmul H
    D'Auria, Anthony C
    Singel, Kelly L
    Davidson, Bruce A
    Knight, Paul R
    Blackwell, Timothy S
    Hohl, Tobias M
    Mansour, Michael K
    Vyas, Jatin M
    Röhm, Marc
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Kelkka, Tiina
    Holmdahl, Rikard
    Segal, Brahm H
    Monocyte- and macrophage-targeted NADPH oxidase mediates antifungal host defense and regulation of acute inflammation in mice2013In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 190, no 8, p. 4175-4184Article in journal (Refereed)
    Abstract [en]

    Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate beta-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation. The Journal of Immunology, 2013, 190: 4175-4184.

  • 17.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Messer, Philipp K
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Stable Redox-Cycling Nitroxide Tempol Inhibits NET Formation2012In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, article id 391Article in journal (Refereed)
    Abstract [en]

    To prevent the spread of pathogens neutrophils as the first line of defense are able to release Neutrophil Extracellular Traps (NETs), a recently discovered form of immune response. Reactive oxygen species (ROS) have been shown to be essential for many different induction routes of NET formation. Therefore, pharmacological inhibition of ROS generation has implications for research and medicine related to NETs. The application of diphenylene iodonium (DPI), an inhibitor of NADPH oxidase activity, is limited due to its toxicity to host cells as well as microbes. Therefore, we investigated the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) a membrane-permeable radical scavenger on NET formation triggered by phorbol esters and Candida albicans. We quantified the amount of NETs with two complementary methods, using a microscopic analysis and an online fluorescence-based assay. In line with removal of ROS, Tempol reduced the amount of NET formation by neutrophils challenged with those stimuli significantly. Since Tempol efficiently blocks NET formation in vitro, it might be promising to test the effect of Tempol in experimental models of disorders in which NETs probably have hazardous effects.

  • 18.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rofougaran, Reza
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Vodnala, Munender
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Kötemann, A
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Adenosine is a drugable negative regulator of neutrophil activity during Candida albicans infectionManuscript (preprint) (Other academic)
  • 19.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lopes, Jose Pedro
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Müller, Daniel C.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Häggman, André
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Holmberg, Sandra
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Grumaz, Christian
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sohn, Kai
    Dieterich, Christoph
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Stable Redox-Cycling Nitroxide Tempol has Antifungal and Immune-modulatory Properties2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1843Article in journal (Refereed)
    Abstract [en]

    Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.

  • 20.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Thompson, Paul R.
    Segal, Brahm H.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nicotine induces neutrophil extracellular traps2016In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 100, no 5, p. 1105-1112Article in journal (Refereed)
    Abstract [en]

    NETs serve to ensnare and kill microbial pathogens. However, NETs can at the same time contribute to tissue damage and excessive inflammation. Nicotine is a major toxic agent and has been associated with exacerbated inflammatory diseases. The current study aimed at investigating the role of nicotine, the addictive component of tobacco and electronic cigarettes, on triggering NET formation. We report that nicotine induces neutrophils to release NETs in a dose-dependent manner. Nicotine-induced NET formation is mediated via nicotine acetylcholine receptors, depends on Akt and PAD4 activation, but is Nox2-independent, as demonstrated by pharmacological inhibition of Nox2 and by use of Nox2-deficient mouse neutrophils. These findings demonstrate that nicotine induces NETs, which may in turn contribute to smoking-related diseases.

  • 21.
    Hosseinzadeh, Ava
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Novel Insight into Neutrophil Immune Responses by Dry Mass Determination of Candida albicans Morphotypes2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, article id e77993Article in journal (Refereed)
    Abstract [en]

    The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI) is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.

  • 22.
    Lopes, Jose Pedro
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Stylianou, Marios
    Backman, Emelie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Holmberg, Sandra
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Jass, Jana
    Claesson, Rolf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Evasion of Immune Surveillance in Low Oxygen Environments Enhances Candida albicans Virulence.2018In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 6, article id e02120-18Article in journal (Refereed)
    Abstract [en]

    Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of β-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic conditions survived. These results suggest that C. albicans benefited from low oxygen niches to increase virulence. The interplay of C. albicans with innate immune cells under these conditions contributed to the overall outcome of infection. Adaption to low oxygen levels was in addition beneficial for C. albicans by reducing susceptibility to selected antifungal drugs. Hence, immunomodulation of host cells under low oxygen conditions could provide a valuable approach to improve current antifungal therapies.

  • 23.
    Lopes, Jose Pedro
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Gunnar
    Stockholm, Sweden.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12287Article in journal (Refereed)
    Abstract [en]

    Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal, colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast cell responses included the launch of initial, intermediate and late phase components determined by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans infection. Consequently, these findings open new doors for understanding fungal pathogenicity.

  • 24.
    Lopes, Jose Pedro
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Visualizing Hypoxia in a Murine Model of Candida albicans Infection Using in vivo Biofluorencence2019In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 15, article id UNSP e3326Article in journal (Refereed)
    Abstract [en]

    Candida albicans is a leading human fungal pathogen that uses several metabolic adaptations to escape immune cells and causes systemic disease. Here, we describe a protocol for measuring one of these adaptations, the ability to thrive in hypoxic niches. Hypoxia was generated after successful subdermal infection with C. albicans in a murine infection model. Hypoxia was measured using a fluorescent dye for carbonic anhydrase 9, a host enzyme active under hypoxic conditions. Emitted fluorescence was subsequently quantified using an IVIS system. This protocol was optimized for the use in subdermal infection in mice but has the potential to be adapted to other models of fungal infection.

  • 25.
    Lopes, José Pedro
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Backman, Emelie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Holmberg, Sandra
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ekoff, Maria
    Nilsson, Gunnar
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Cryptococcus neoformans Induces MCP-1 Release and Delays the Death of Human Mast Cells2019In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 289Article in journal (Refereed)
    Abstract [en]

    Cryptococcosis, caused by the basidiomycete Cryptococcus neoformans, is a life-threatening disease affecting approximately one million people per year worldwide. Infection can occur when C. neoformans cells are inhaled by immunocompromised people. In order to establish infection, the yeast must bypass recognition and clearance by immune cells guarding the tissue. Using in vitro infections, we characterized the role of mast cells (MCs) in cryptococcosis. We found that MCs recognize C. neoformans and release inflammatory mediators such as tryptase and cytokines. From the latter group MCs released mainly CCL-2/MCP-1, a strong chemoattractant for monocytic cells. We demonstrated that supernatants of infected MCs recruit monocytes but not neutrophils. During infection with C. neoformans, MCs have a limited ability to kill the yeast depending on the serotype. C. neoformans, in turn, modulates the lifespan of MCs both, by presence of its polysaccharide capsule and by secreting soluble modulators. Taken together, MCs might have important contributions to fungal clearance during early stages of cryptocococis where these cells regulate recruitment of monocytes to mucosal tissues.

  • 26. Loures, Flávio V
    et al.
    Röhm, Marc
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lee, Chrono K
    Santos, Evelyn
    Wang, Jennifer P
    Specht, Charles A
    Calich, Vera L G
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Levitz, Stuart M
    Recognition of Aspergillus fumigatus Hyphae by Human Plasmacytoid Dendritic Cells Is Mediated by Dectin-2 and Results in Formation of Extracellular Traps2015In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, no 2, article id e1004643Article in journal (Refereed)
    Abstract [en]

    Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.

  • 27.
    Nelson, Maria
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Niemic, Maria Joanna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nahrevanian, Shahab
    Urban, Constantin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Malaria-enhanced Neutrophil Dependent Clearance of S. pneumoniae in an in vivo Co-infection ModelManuscript (preprint) (Other (popular science, discussion, etc.))
  • 28.
    Niemiec, Maria Joanna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    De Samber, B.
    Garrevoet, J.
    Vergucht, E.
    Vekemans, B.
    De Rycke, R.
    Björn, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wellenreuther, G.
    Falkenberg, G.
    Cloetens, P.
    Vincze, L.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Trace element landscape of resting and activated human neutrophils on the sub-micrometer level2015In: Metallomics, ISSN 1756-591X, Vol. 7, no 6, p. 996-1010Article in journal (Refereed)
    Abstract [en]

    Every infection is a battle for trace elements. Neutrophils migrate first to the infection site and accumulate quickly to high numbers. They fight pathogens by phagocytosis and intracellular toxication. Additionally, neutrophils form neutrophil extracellular traps (NETs) to inhibit extracellular microbes. Yet, neutrophil trace element characteristics are largely unexplored. We investigated unstimulated and phorbol myristate acetate-stimulated neutrophils using synchrotron radiation X-ray fluorescence (SR-XRF) on the sub-micron spatial resolution level. PMA activates pinocytosis, cytoskeletal rearrangements and the release of NETs, all mechanisms deployed by neutrophils to combat infection. By analyzing Zn, Fe, Cu, Mn, P, S, and Ca, not only the nucleus but also vesicular granules were identifiable in the elemental maps. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) revealed a neutrophil-specific composition of Zn, Fe, Cu, and Mn in comparison with J774 and HeLa cells, indicating a neutrophil-specific metallome complying with their designated functions. When investigating PMA-activated neutrophils, the SR-XRF analysis depicted typical subcellular morphological changes: the transformation of nucleus and granules and the emergence of void vacuoles. Mature NETs were evenly composed of Fe, P, S, and Ca with occasional hot spots containing Zn, Fe, and Ca. An ICP-MS-based quantification of NET supernatants revealed a NETosis-induced decrease of soluble Zn, whereas Fe, Cu, and Mn concentrations were only slightly affected. In summary, we present a combination of SR-XRF and ICP-MS as a powerful tool to analyze trace elements in human neutrophils. The approach will be applicable and valuable to numerous aspects of nutritional immunity.

  • 29.
    Niemiec, Maria Joanna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). HKI, Leibniz Inst Nat Product Res & Infect Biol, Jena, Germany.
    Grumaz, Christian
    Ermert, David
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Lund Univ, Div Med Prot Chem, Dept Translat Med, Malmo, Sweden.
    Desel, Christiane
    Shankar, Madhu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lopes, Jose Pedro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Mills, Ian G.
    Stevens, Philip
    Sohn, Kai
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Dual transcriptome of the immediate neutrophil and Candida albicans interplay2017In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 18, article id 696Article in journal (Refereed)
    Abstract [en]

    Background: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. Methods: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. Results: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil-and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. Conclusions: Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.

  • 30.
    Röhm, Marc
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Grimm, Melissa J.
    D'Auria, Anthony C.
    Almyroudis, Nikolaos G.
    Segal, Brahm H.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    NADPH Oxidase Promotes Neutrophil Extracellular Trap Formation in Pulmonary Aspergillosis2014In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 5, p. 1766-1777Article in journal (Refereed)
    Abstract [en]

    NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wildtype and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.

  • 31.
    Röhm, Marc
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Lindemann, E.
    Hiller, E.
    Ermert, David
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Lemuth, K.
    Trkulja, D.
    Sogukpinar, O.
    Brunner, H.
    Rupp, S.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sohn, K.
    A family of secreted pathogenesis-related proteins in Candida albicans2013In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 87, no 1, p. 132-151Article in journal (Refereed)
    Abstract [en]

    Analysing culture supernatants of yeast and hyphal cells of Candida albicans, we found two close homologues of pathogenesis-related (PR-) 1 proteins, Rbe1p and Rbt4p, in the secretome. Due to sequence homology, three additional, yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, together with RBE1 and RBT4 were assigned to a novel family of CaPRY proteins. In a Δrbe1/Δrbt4 deletion strain, genome-wide transcriptional analysis revealed differential transcription of only a limited set of genes implicated in virulence and oxidative stress response. Single deletion of RBE1 or RBT4 in a clinical C.albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in a Δrbe1/Δrbt4 double deletion strain, where virulence was strongly affected. Remarkably, transcription of RBT4 and RBE1 was each upregulated in blastospores of Δrbe1 or hyphae of Δrbt4 deletion strains respectively, indicating functional complementation thereby compensating a potential virulence defect in the single deletion strains. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leucocytes. Therefore, the crucial contribution of both C.albicans pathogenesis-related proteins to virulence might be vested in protection against phagocyte attack.

  • 32.
    Seper, Andrea
    et al.
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gorkiewicz, Gregor
    University of Graz, Graz, Austria.
    Lichtenegger, Sabine
    University of Graz, Graz, Austria.
    Roier, Sandro
    University of Graz, Graz, Austria.
    Leitner, Deborah R
    University of Graz, Graz, Austria.
    Röhm, Marc
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Grutsch, Andreas
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Reidl, Joachim
    University of Graz, Graz, Austria.
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Schild, Stefan
    Institute of Molecular Biosciences, University of Graz, Graz, Austria.
    Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 9, article id e1003614Article in journal (Refereed)
    Abstract [en]

    The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  • 33. Singel, Kelly L.
    et al.
    Grzankowski, Kassondra S.
    Khan, A. N. M. Nazmul H.
    Grimm, Melissa J.
    D'Auria, Anthony C.
    Morrell, Kayla
    Eng, Kevin H.
    Hylander, Bonnie
    Mayor, Paul C.
    Emmons, Tiffany R.
    Lénárt, Nikolett
    Fekete, Rebeka
    Környei, Zsuzsanna
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Itagaki, Kiyoshi
    Hauser, Carl J.
    Leifer, Cynthia
    Moysich, Kirsten B.
    Odunsi, Kunle
    Dénes, Ádám
    Segal, Brahm H.
    Mitochondrial DNA in the tumour microenvironment activates neutrophils and is associated with worse outcomes in patients with advanced epithelial ovarian cancer2019In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 120, no 2, p. 207-217Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes.

    METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE.

    RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02).

    CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.

  • 34. Singel, Kelly L.
    et al.
    Grzankowski, Kassondra S.
    Khan, ANM Nazmul H.
    Grimm, Melissa J.
    D’Auria, Anthony C.
    Morrell, Kayla
    Eng, Kevin H.
    Hylander, Bonnie
    Mayor, Paul C.
    Emmons, Tiffany R.
    Lénárt, Nikolett
    Fekete, Rebeka
    Környei, Zsuzsanna
    Muthukrishnan, Uma
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Itagaki, Kiyoshi
    Hauser, Carl J.
    Leifer, Cynthia
    Moysich, Kirsten B.
    Odunsi, Kunle
    Dénes, Ádám
    Segal, Brahm H.
    Mitochondrial DNA in the tumor microenvironment activates neutrophils and is associated with worsened outcomes in patients with advanced epithelial ovarian cancerManuscript (preprint) (Other academic)
    Abstract [en]

    Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesized that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worsened outcomes.Banked ascites supernatants from patients with newly diagnosed advanced EOC were analyzed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELPand ELANE. The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n=68, log-rank, p=0.0178). NETs were detected in resected tumors. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivostimulated T cell proliferation. Increased SELPmRNA expression correlated with worsened overall survival (n=302, Cox model, p=0.02).In this single-center retrospective analysis, ascites mtDNA correlated with worsened PFS in advanced EOC. Our results support mitochondrial and other DAMPs activating neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumor immunity. These pathways are potential prognostic markers and therapeutic targets.

  • 35. Sohn, K
    et al.
    Roehm, M
    Urban, Constantin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saunders, N
    Rothenstein, D
    Lottspeich, F
    Schröppel, K
    Brunner, H
    Rupp, S
    Identification and characterization of Cor33p, a novel protein implicated in tolerance towards oxidative stress in Candida albicans2005In: Eukaryotic Cell, ISSN 1535-9778, E-ISSN 1535-9786, Vol. 4, no 12, p. 2160-2169Article in journal (Refereed)
    Abstract [en]

    We applied two-dimensional gel electrophoresis to identify downstream effectors of CPH1 and EFG1 under hypha-inducing conditions in Candida albicans. Among the proteins that were expressed in wild-type cells but were strongly downregulated in a cph1Delta/efg1Delta double mutant in alpha-minimal essential medium at 37 degrees C, we could identify not-yet-characterized proteins, including Cor33-1p and Cor33-2p. The two proteins are almost identical (97% identity) and represent products of allelic isoforms of the same gene. Cor33p is highly similar to Cip1p from Candida sp. but lacks any significant homology to proteins from Saccharomyces cerevisiae. Strikingly, both proteins share homology with phenylcoumaran benzylic ether reductases and isoflavone reductases from plants. For other hypha-inducing media, like yeast-peptone-dextrose (YPD) plus serum at 37 degrees C, we could not detect any transcription for COR33 in wild-type cells, indicating that Cor33p is not hypha specific. In contrast, we found a strong induction for COR33 when cells were treated with 5 mM hydrogen peroxide. However, under oxidative conditions, transcription of COR33 was not dependent on EFG1, indicating that other regulatory factors are involved. In fact, upregulation depends on CAP1 at least, as transcript levels were clearly reduced in a Deltacap1 mutant strain under oxidative conditions. Unlike in wild-type cells, transcription of COR33 in a tsa1Delta mutant can be induced by treatment with 0.1 mM hydrogen peroxide. This suggests a functional link between COR33 and thiol-specific antioxidant-like proteins that are important in the oxidative-stress response in yeasts. Concordantly, cor33Delta deletion mutants show retarded growth on YPD plates supplemented with hydrogen peroxide, indicating that COR33 in general is implicated in conferring tolerance toward oxidative stress on Candida albicans.

  • 36. Sohn, K
    et al.
    Urban, Constantin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Brunner, H
    Rupp, S
    EFG1 is a major regulator of cell wall dynamics in Candida albicans as revealed by DNA microarrays2003In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 47, no 1, p. 89-102Article in journal (Refereed)
    Abstract [en]

    Cell wall dynamics in Candida albicans, the most common fungal pathogen in man, underlie regulatory processes during the yeast-to-hyphae transition. To analyse this regulation at the transcriptional level, we have established a DNA microarray representing genes implicated in cell wall biogenesis. Using these microarrays, we were able to identify YWP1 and HWP2 that are specifically transcribed in the yeast or hyphal growth form respectively. Cluster analysis revealed at least two major clusters of genes: cluster I comprised genes that were upregulated under at least one hyphae-inducing condition. Three as yet not further characterized genes were attributed to cluster II. These genes were transcribed in the yeast form of C. albicans and were downregulated in an EFG1-dependent manner under specific hyphae-inducing conditions. We show further that, in contrast to CPH1, EFG1 plays a major role in the transcriptional regulation of cell wall proteins under the conditions investigated. EFG1 was essential for the transcription of both hyphae-specific genes such as HWP1 and HWP2 as well as the yeast form-specific gene YWP1. Moreover, we found that, under various conditions, EFG1 also can act as a strong repressor for the transcription of RBE1, another not yet characterized cell wall protein. Overall, our data show that EFG1 plays a major role in the induction and repression of cell wall genes, not only in the hyphal form but also in the yeast form of C. albicans.

  • 37. Sohn, Kai
    et al.
    Schwenk, Jochen
    Urban, Constantin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lechner, Johannes
    Schweikert, Michael
    Rupp, Steffen
    Getting in touch with Candida albicans: the cell wall of a fungal pathogen2006In: Current Drug Targets, ISSN 1389-4501, E-ISSN 1873-5592, Vol. 7, no 4, p. 505-512Article in journal (Refereed)
    Abstract [en]

    The cell wall of fungi is a highly complex structure consisting of a network of polysaccharides in which a plethora of different proteins are embedded. It is one of the major organelles of the cell surrounding it like an armor which protects from environmental stresses like osmotic pressure and defines the shape and physical strength of the fungal cell. It is crucial for colonization and infection since it defines the interface between host and pathogen. No similar structure is present in the host, therefore it defines a prime target for drug development. In this context, it has been shown that cell surface proteins are required for adhesion to host cells. The fact, that both pathogenic fungi, like Candida albicans as well as non-pathogenic fungi, like Saccharomyces cerevisiae, in general, have a very similar polysaccharide structure but differ significantly in their protein composition which underscores the importance of cell wall proteins for pathogenesis. However, cell wall proteomics of fungi is a highly challenging task due to the complex biochemistry of these proteins. The extensive post-translational modifications and covalent attachment to the polysaccharide backbone of a large proportion of cell wall proteins makes it a demanding task to isolate and identify them. In this article, we describe the recent approaches that have been developed to describe cell wall dynamics and to isolate and identify cell wall proteins in the pathogenic yeast C. albicans.

  • 38.
    Stylianou, Marios
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Kulesskiy, Evgeny
    Lopes, José Pedro
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Wennerberg, Krister
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Antifungal Application of Nonantifungal Drugs2014In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 58, no 2, p. 1055-1062Article in journal (Refereed)
    Abstract [en]

    Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to similar to 150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.

  • 39.
    Stylianou, Marios
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Uvell, Hanna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lopes, Jose Pedro
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Enquist, Per-Anders
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Elofsson, Mikael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Novel High-Throughput Screening Method for Identification of Fungal Dimorphism Blockers2015In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 20, no 2, p. 285-91Article in journal (Refereed)
    Abstract [en]

    Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus, morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably, Z' factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal. Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.

  • 40.
    Thorslund, Sara E
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ermert, D
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Urban, Constantin
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Role of YopK in Yersinia resistance against polymorphonuclear leukocyte defenseManuscript (preprint) (Other academic)
  • 41.
    Thorslund, Sara E
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ermert, David
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Erttmann, Saskia F
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Role of YopK in Yersinia pseudotuberculosis Resistance Against Polymorphonuclear Leukocyte Defense2013In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 1, p. 11-22Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Y. pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type three secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte (PMN, or neutrophil) defense. A yopK mutant, which is attenuated in the mouse infection model where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and ROS production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction PMN death, whereas the mutant caused a necrotic-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism where YopK functions to prevent unintended Yop delivery and thereby PMN disruption resulting in necrotic like cell death, which would enhance the inflammatory response favoring the host.

  • 42.
    Thunström Salzer, Anna
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Niemiec, Maria Joanna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Stylianou, Marios
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Åstrom, Fredrik
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Röhm, Marc
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Wahlin, Anders
    Umeå University, Faculty of Medicine, Department of Radiation Sciences.
    Ermert, David
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Urban, Constantin F.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients2018In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 9, article id 1968Article in journal (Refereed)
    Abstract [en]

    Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.

  • 43.
    Urban, Constantin F
    et al.
    Department for Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Ermert, David
    Schmid, Monika
    Abu-Abed, Ulrike
    Goosmann, Christian
    Nacken, Wolfgang
    Brinkmann, Volker
    Jungblut, Peter R
    Zychlinsky, Arturo
    Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans2009In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 5, no 10, article id e1000639Article in journal (Refereed)
    Abstract [en]

    Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

  • 44.
    Urban, Constantin F
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lourido, Sebastian
    Zychlinsky, Arturo
    How do microbes evade neutrophil killing?2006In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, no 11, p. 1687-1696Article in journal (Refereed)
    Abstract [en]

    Many microbial pathogens evolved to circumvent the attack of neutrophils, which are essential effector cells of the innate immune system. Here we review six major strategies that pathogenic bacteria and fungi use to evade neutrophil defences: (i) turning on survival and stress responses, (ii) avoiding contact, (iii) preventing phagocytosis, (iv) surviving intracellularly, (v) inducing cell death and (vi) evading killing by neutrophil extracellular traps. For each category we give examples and further focus on one particular pathogenic microbe in more detail. Pathogens include Candida albicans, Cryptococcus neoformans, Yersinia ssp., Helicobacter pylori, Staphylococcus aureus, Streptococcus pyogenes and Streptococcus pneumoniae.

  • 45.
    Urban, Constantin F.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nett, Jeniel E
    Neutrophil extracellular traps in fungal infection2019In: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 89, p. 47-57Article in journal (Refereed)
    Abstract [en]

    Fungal infections are a continuously increasing problem in modern health care. Understanding the complex biology of the emerging pathogens and unraveling the mechanisms of host defense may form the basis for the development of more efficient diagnostic and therapeutic tools. Neutrophils play a pivotal role in the defense against fungal pathogens. These phagocytic hunters migrate towards invading fungal microorganisms and eradicate them by phagocytosis, oxidative burst and release of neutrophil extracellular traps (NETs). In the last decade, the process of NET formation has received unparalleled attention, with numerous studies revealing the relevance of this neutrophil function for control of various mycoses. Here, we describe NET formation and summarize its role as part of the innate immune defense against fungal pathogens. We highlight factors influencing the formation of these structures and molecular mechanisms employed by fungi to impair the formation of NETs or subvert their antifungal effects.

  • 46.
    Urban, Constantin F
    et al.
    Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Schumannstrasse 21/22, 10117 Berlin, Germany.
    Reichard, Ulrike
    Brinkmann, Volker
    Zychlinsky, Arturo
    Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms2006In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, no 4, p. 668-676Article in journal (Refereed)
    Abstract [en]

    Neutrophils phagocytose and kill microbes upon phagolysosomal fusion. Recently we found that activated neutrophils form extracellular fibres that consist of granule proteins and chromatin. These neutrophil extracellular traps (NETs) degrade virulence factors and kill Gram positive and negative bacteria. Here we show for the first time that Candida albicans, a eukaryotic pathogen, induces NET-formation and is susceptible to NET-mediated killing. C. albicans is the predominant aetiologic agent of fungal infections in humans, particularly in immunocompromised hosts. One major virulence trait of C. albicans is its ability to reversibly switch from singular budding cells to filamentous hyphae. We demonstrate that NETs kill both yeast-form and hyphal cells, and that granule components mediate fungal killing. Taken together our data indicate that neutrophils trap and kill ascomycetous yeasts by forming NETs.

  • 47.
    Urban, Constantin F.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Reichenbach, J.
    Bianchi, M.
    Niemiec, Maria Joanna
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Neutrophil extracellular traps in opportunistic mycoses2012In: Mycoses (Berlin), ISSN 0933-7407, E-ISSN 1439-0507, Vol. 55, no Supplement 4, p. 24-24Article in journal (Refereed)
  • 48. Urban, Constantin
    et al.
    Sohn, K
    Lottspeich, F
    Brunner, H
    Rupp, S
    Identification of cell surface determinants in Candida albicans reveals Tsa1p, a protein differentially localized in the cell2003In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 544, no 1-3, p. 228-235Article in journal (Refereed)
    Abstract [en]

    To identify cell surface proteins of Candida albicans, the predominant fungal pathogen in humans, we have established an approach using a membrane impermeable biotin derivative in combination with affinity purification. We were able to identify 29 different proteins under two distinct conditions. Among mannoproteins, heat shock proteins and glycolytic enzymes we found thiol-specific antioxidant-like protein 1 (Tsa1p) to be differentially localized depending on the conditions applied. Only in hyphally grown cells Tsa1p was localized to the cell surface whereas in blastospores no surface but mainly nuclear localization was found. This indicates that cell surface expression of at least some proteins is mediated by differential translocation.

  • 49.
    Urban, Constantin
    et al.
    Fraunhofer IGB, 70569 Stuttgart, Germany.
    Xiong, Xin
    Sohn, Kai
    Schröppel, Klaus
    Brunner, Herwig
    Rupp, Steffen
    The moonlighting protein Tsa1p is implicated in oxidative stress response and in cell wall biogenesis in Candida albicans2005In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 57, no 5, p. 1318-1341Article in journal (Refereed)
    Abstract [en]

    Candida albicans is one of the most common fungal pathogens in humans. The cell wall is the first contact site between host and pathogen and thus is critical for colonization and infection of the host. We have identified Tsa1p, a protein that is differentially localized to the cell wall of C. albicans in hyphal cells but remains in the cytosol and nucleus in yeast-form cells. This is different from Saccharomyces cerevisiae, where the homologous protein solely has been found in the cytoplasm. We report here that TSA1 confers resistance towards oxidative stress as well as is involved in the correct composition of hyphal cell walls. However, no significant change of the cell wall composition was observed in a TSA1 deletion strain in yeast-form cells, which is in good agreement with the observation that Tsa1p is absent from the yeast-form cell wall. This indicates that Tsa1p of C. albicans might represent a moonlighting protein with specific functions correlating to its respective localization. Furthermore, the translocation of Tsa1p to the hyphal cell wall of C. albicans depends on Efg1p, suggesting a contribution of the cAMP/PKA pathway to the localization of this protein. In a strain deleted for TUP1 that filaments constitutively Tsa1p can be found in the cell wall under all conditions tested, confirming the result that Tsa1p localization to the cell wall is correlated to the morphology of C. albicans.

  • 50.
    Urban, Constantin
    et al.
    Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Charitéplatz 1, Berlin 10117, Germany.
    Zychlinsky, Arturo
    Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Charitéplatz 1, Berlin 10117, Germany.
    Netting bacteria in sepsis2007In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 13, no 4, p. 403-404Article in journal (Other academic)
12 1 - 50 of 51
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