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Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

The peptide toxin candidalysin, secreted by Candida albicans hyphae, promotes stimulation of neutrophil extracellular traps (NETs). However, candidalysin alone triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leucotoxic hypercitrullination, which is governed by calcium-induced protein arginine deaminase 4 activation and initiation of intracellular signalling events in a dose- and time-dependent manner. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Candidalysin-triggered NLS demonstrate anti-Candida activity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+. This study reveals that C. albicans hyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entangles C. albicans hyphae and efficiently inhibits their growth.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Interaction between microorganisms and their surroundings are generally mediated via the cell wall or cell envelope. An understanding of the overall chemical composition of these surface layers may give clues on how these interactions occur and suggest mechanisms to manipulate them. This knowledge is key, for instance, in research aiming to reduce colonization of medical devices and device-related infections from different types of microorganisms. In this context, X-ray photoelectron spectroscopy (XPS) is a powerful technique as its analysis depth below 10Â nm enables studies of the outermost surface structures of microorganism. Of specific interest for the study of biological systems is cryogenic XPS (cryo-XPS). This technique allows studies of intact fast-frozen hydrated samples without the need for pre-treatment procedures that may cause the cell structure to collapse or change due to the loss of water. Previously, cryo-XPS has been applied to study bacterial and algal surfaces with respect to their composition of lipids, polysaccharides and peptide (protein and/or peptidoglycan). This contribution focuses onto two other groups of microorganisms with widely different architecture and modes of life, namely fungi and viruses. It evaluates to what extent existing models for data treatment of XPS spectra can be applied to understand the chemical composition of their very different surface layers. XPS data from model organisms as well as reference substances representing specific building blocks of their surface were collected and are presented. These results aims to guide future analysis of the surface chemical composition of biological systems.

Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.

Opportunistic, invasive mycoses in immunocompromised patients remain challenging for health care with unacceptably high levels of morbidity and mortality. Neutrophils are essential in host protection against invasive mycoses. Upon development of acute infection, neutrophils are recruited from circulation to the infected tissue, where they exert a considerable variety of effector functions with the ultimate task to eradicate invading microbes. Effector functions include recognition, phagocytosis and intracellular killing of microorganisms via oxidative and non-oxidative mechanisms, excretion of antimicrobial factors from intracellular storages (degranulation), release of neutrophil extracellular traps (NETs) and of extracellular vesicles (EVs), as well as generation of cytokines and chemokines to modulate immune responses. Herein, we describe recent findings which further our understanding of the roles of neutrophils during opportunistic fungal infections which could serve as starting point for the development of immune-targeted interventions to improve clinical management of affected individuals.

Analytical capabilities of Nanoscopic Secondary Ion Mass Spectrometry (nano-SIMS) and Synchrotron Radiation based X-ray Fluorescence (SR nano-XRF) techniques were compared for nanochemical imaging of polymorphonuclear human neutrophils (PMNs). PMNs were high pressure frozen (HPF), cryosubstituted, embedded in Spurr's resin and cut in thin sections (500 nm and 2 mu m for both techniques resp.) Nano-SIMS enabled nanoscale mapping of isotopes of C, N, O, P and S, while SR based nano-XRF enabled trace level imaging of metals like Ca, Mn, Fe, Ni, Cu and Zn at a resolution of approx. 50 nm. The obtained elemental distributions were compared with those of whole, cryofrozen PMNs measured at the newly developed ID16A nano-imaging beamline at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Similarities were observed for elements more tightly bound to the cell structure such as phosphorus and sulphur, while differences for mobile ions such as chlorine and potassium were more pronounced. Due to the observed elemental redistribution of mobile ions such as potassium and chlorine, elemental analysis of high pressure frozen (HPF), cryo-substituted and imbedded cells should be interpreted critically. Although decreasing analytical sensitivity occurs due to the presence of ice, analysis of cryofrozen cells - close to their native state - remains the golden standard. In general, we found nanoscale secondary ion mass spectrometry (nano-SIMS) and synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF) to be two supplementary alternatives for nanochemical imaging of single cells at the nanoscale.

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., Originally included in thesis in manuscript form

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.

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.

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.

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 condi

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.

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., Funding Agencies:Cancer Research Foundation in Northern Sweden AMP 11684 LP 10-1833 Västerbotten County Council (VLL-ALF) Laboratory for Molecular Infection Medicine Sweden (MIMS) Medical Faculty Umeå University 316-886-10

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.

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.

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., Originally published in manuscript form with title [RNA-Seq transcription profile of the neutrophil: Candida albicans in vitro interaction]Errata BMC Genomics (2017) 18:696 DOI: 10.1186/s12864-017-4097-4