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Background: Interleukin (IL)-1beta has an important role in antifungal defense mechanisms. The inflammasome is thought to be required for caspase-1 activation and processing of the inactive precursor pro-IL-1beta. The aim of the present study was to investigate the pathways of IL-1beta production induced by Candida albicans in human monocytes. Methods: Human mononuclear cells were stimulated with C. albicans or mutant strains defective in mannosylation or chitin. Receptors were blocked with specific antagonists, and the IL-1beta concentration was measured. Results: Human primary monocytes produce bioactive IL-1beta when stimulated with C. albicans. The transcription of IL-1beta was induced through mannose receptor (MR), Toll-like receptor (TLR) 2, and dectin-1 but not through TLR4 and TLR9. N-mannan-linked residues, chitin, and beta-glucan from C. albicans are important for IL-1beta stimulation. Surprisingly, processing and secretion of IL-1beta in monocytes did not require pathogen-mediated inflammasome activation, because of the constitutive activation of caspase-1 and the capability of monocytes to release endogenous adenosine-5'-triphosphate. Conclusions: This study is the first dissection of the molecular mechanisms of IL-1beta production by a fungal pathogen. Transcription through mannan/chitin/MR and beta-glucan/dectin-1/TLR2 induces production of IL-1beta by C. albicans in human monocytes, whereas processing of IL-1beta is mediated by constitutively active caspase-1. [ABSTRACT FROM AUTHOR]

Importance: Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans , for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail., Competing Interests: The authors declare no conflict of interest.

The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host's arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival., Importance: The availability and metabolism of amino acids are increasingly recognized as crucial regulators of immune functions. In acute infections, the conversion of the "conditionally essential" amino acid l-arginine by the inducible nitric oxide synthase to nitric oxide is a resistance factor that is produced by the host to fight pathogens. Manipulation of these host defense mechanisms by the pathogen can be key to successful host invasion. We show here that the human opportunistic fungal pathogen Candida albicans influences l-arginine availability for nitric oxide production by induction of the substrate-competing host enzyme arginase-1. This led to a reduced production of nitric oxide and, moreover, reduced eradication of the fungus by human macrophages. We demonstrate that blocking of host arginase-1 activity restored nitric oxide production and increased the killing potential of macrophages. These results highlight the therapeutic potential of l-arginine metabolism in fungal diseases., (Copyright © 2017 Wagener et al.)

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

Fungi cause more than a billion skin infections, more than 100 million mucosal infections, 10 million serious allergies and more than a million deaths each year. Global mortality owing to fungal infections is greater than for malaria and breast cancer and is equivalent to that owing to tuberculosis (TB) and HIV. These statistics evidence fungal infections as a major threat to human health and a major burden to healthcare budgets worldwide. Those patients who are at greatest risk of life-threatening fungal infections include those who have weakened immunity or have suffered trauma or other predisposing infections such as HIV. To address these global threats to human health, more research is urgently needed to understand the immunopathology of fungal disease and human disease susceptibility in order to augment the advances being made in fungal diagnostics and drug development. Here, we highlight some recent advances in basic research in medical mycology and fungal immunology that are beginning to inform clinical decisions and options for personalized medicine, vaccine development and adjunct immunotherapies.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'., (© 2016 The Authors.)

Emerging infections caused by fungi have become a widely recognized global phenomenon. Their notoriety stems from their causing plagues and famines, driving species extinctions, and the difficulty in treating human mycoses alongside the increase of their resistance to antifungal drugs. This special issue comprises a collection of articles resulting from a Royal Society discussion meeting examining why pathogenic fungi are causing more disease now than they did in the past, and how we can tackle this rapidly emerging threat to the health of plants and animals worldwide.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'., (© 2016 The Author(s).)

Candida albicans is a commensal coloniser of most people and a pathogen of the immunocompromised or patients in which barriers that prevent dissemination have been disrupted. Both the commensal and pathogenic states involve regulation and adaptation to the host microenvironment. The pathogenic potential can be downregulated to sustain commensalism or upregulated to damage host tissue and avoid and subvert immune surveillance. In either case it seems as though the cell biology of this fungus has evolved to enable the establishment of different types of relationships with the human host. Here we summarise latest advances in the analysis of mechanisms that enable C. albicans to occupy different body sites whilst avoiding being eliminated by the sentinel activities of the human immune system., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies., Competing Interests: The authors declare no competing financial interests.

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.

Despite the fact that Candida albicans is an important human fungal pathogen and Dectin-2 is a major pattern recognition receptor for fungi, our knowledge regarding the role of Dectin-2 for the host defense against disseminated candidiasis is limited. Dectin-2 deficient (Dectin-2(-/-)) mice were more susceptible to systemic candidiasis, and the susceptibility was mirrored by an elevated fungal load in the kidneys that correlated with the presence of large inflammatory foci. Phagocytosis of Candida by the macrophages lacking the Dectin-2 receptor was moderately decreased, while production of most of the macrophage-derived cytokines from Dectin-2(-/-) mice with systemic candidiasis was decreased. No striking differences among several Candida mutants defective in mannans could be detected between naïve wild-type and Dectin-2(-/-) mice, apart from the β-mannan-deficient bmt1Δ/bmt2Δ/bmt5Δ triple mutant, suggesting that β-mannan may partially mask α-mannan detection, which is the major fungal structure recognized by Dectin-2. Deciphering the mechanisms responsible for host defense against the majority of C. albicans strains represents an important step in understanding the pathophysiology of systemic candidiasis, which might lead to the development of novel immunotherapeutic strategies.

The surveillance and elimination of fungal pathogens rely heavily on the sentinel behaviour of phagocytic cells of the innate immune system, especially macrophages and neutrophils. The efficiency by which these cells recognize, uptake and kill fungal pathogens depends on the size, shape and composition of the fungal cells and the success or failure of various fungal mechanisms of immune evasion. In this Review, we describe how fungi, particularly Candida albicans, interact with phagocytic cells and discuss the many factors that contribute to fungal immune evasion and prevent host elimination of these pathogenic microorganisms.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency disorder characterised by susceptibility to chronic Candida and fungal dermatophyte infections of the skin, nails and mucous membranes. Molecular epidemiology studies of CMC infection are limited in number and scope and it is not clear whether single or multiple strains inducing CMC persist stably or are exchanged and replaced. We subjected 42 C. albicans individual single colony isolates from 6 unrelated CMC patients to multilocus sequence typing (MLST). Multiple colonies were typed from swabs taken from multiple body sites across multiple time points over a 17-month period. Among isolates from each individual patient, our data show clonal and persistent diploid sequence types (DSTs) that were stable over time, identical between multiple infection sites and exhibit azole resistant phenotypes. No shared origin or common source of infection was identified among isolates from these patients. Additionally, we performed C. albicans MLST SNP genotype frequency analysis to identify signatures of past loss of heterozygosity (LOH) events among persistent and azole resistant isolates retrieved from patients with autoimmune disorders including CMC.

Pathogenic and non-pathogenic fungi synthesize glycosphingolipids, which have a crucial role in growth and viability. Glycosphingolipids also contribute to fungal-associated pathogenesis. The opportunistic yeast pathogen Candida albicans synthesizes phospholipomannan (PLM), which is a glycosphingolipid of the mannosylinositol phosphorylceramide family. Through its lipid and glycan moieties, PLM contributes to the initial recognition of the yeast, causing immune system disorder and persistent fungal disease through activation of host signaling pathways. The lipid moiety of PLM activates the deregulation signaling pathway involved in yeast phagocytosis whereas its glycan moiety, composed of β-1,2 mannosides (β-Mans), participates to inflammatory processes through a mechanism involving Galectin-3. Biosynthesis of PLM β-Mans involves two β-1,2 mannosyltransferases (Bmts) that initiate (Bmt5) and elongate (Bmt6) the glycan chains. After generation of double bmtsΔ mutants, we show that Bmt5 has redundant activity with Bmt2, which can replace Bmt5 in bmt5Δ mutant. We also report that PLM is located in the inner layer of the yeast cell wall. PLM seems to be not essential for systemic infection of the yeast. However, defect of PLM β-mannosylation increases resistance of C. albicans to inhibitors of β-glucans and chitin synthesis, highlighting a role of PLM in cell wall homeostasis., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall β-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections., (Copyright © 2015, Walker et al.)

The major fungal pathogen of humans, Candida albicans, mounts robust responses to oxidative stress that are critical for its virulence. These responses counteract the reactive oxygen species (ROS) that are generated by host immune cells in an attempt to kill the invading fungus. Knowledge of the dynamical processes that instigate C. albicans oxidative stress responses is required for a proper understanding of fungus-host interactions. Therefore, we have adopted an interdisciplinary approach to explore the dynamical responses of C. albicans to hydrogen peroxide (H2O2). Our deterministic mathematical model integrates two major oxidative stress signalling pathways (Cap1 and Hog1 pathways) with the three major antioxidant systems (catalase, glutathione and thioredoxin systems) and the pentose phosphate pathway, which provides reducing equivalents required for oxidative stress adaptation. The model encapsulates existing knowledge of these systems with new genomic, proteomic, transcriptomic, molecular and cellular datasets. Our integrative approach predicts the existence of alternative states for the key regulators Cap1 and Hog1, thereby suggesting novel regulatory behaviours during oxidative stress. The model reproduces both existing and new experimental observations under a variety of scenarios. Time- and dose-dependent predictions of the oxidative stress responses for both wild type and mutant cells have highlighted the different temporal contributions of the various antioxidant systems during oxidative stress adaptation, indicating that catalase plays a critical role immediately following stress imposition. This is the first model to encapsulate the dynamics of the transcriptional response alongside the redox kinetics of the major antioxidant systems during H2O2 stress in C. albicans.

β-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of β-1,2-oligomannosides (β-Mans) to phosphopeptidomannan and phospholipomannan. β-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor β-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with β-Mans but only at the nonreducing end of α-1,2-mannotriose. β-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 β-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for β-1,2-mannosylation of CWMPs and expression of β-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no β-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the β-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock., Importance: The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock., (Copyright © 2015 Ene et al.)

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans., (Copyright © 2015, Chavez-Dozal et al.)

The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3⎔ cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.

The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild-type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease., (© 2014 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Avoidance of innate immune defense is an important mechanism contributing to the pathogenicity of microorganisms. The fungal pathogen Candida albicans undergoes morphogenetic switching from the yeast to the filamentous hyphal form following phagocytosis by macrophages, facilitating its escape from the phagosome, which can result in host cell lysis. We show that the intracellular host trafficking GTPase Rab14 plays an important role in protecting macrophages from lysis mediated by C. albicans hyphae. Live-cell imaging of macrophages expressing green fluorescent protein (GFP)-tagged Rab14 or dominant negative Rab14, or with small interfering RNA (siRNA)-mediated knockdown of Rab14, revealed the temporal dynamics of this protein and its influence on the maturation of macrophage phagosomes following the engulfment of C. albicans cells. Phagosomes containing live C. albicans cells became transiently Rab14 positive within 2 min following engulfment. The duration of Rab14 retention on phagosomes was prolonged for hyphal cargo and was directly proportional to hyphal length. Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing. However, Rab14 depletion delayed the acquisition of the late phagosome maturation markers LAMP1 and lysosomal cathepsin, indicating delayed formation of a fully bioactive lysosome. This was associated with a significant increase in the level of macrophage killing by C. albicans. Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape., (Copyright © 2015 Okai et al.)

The theoretical physicist and Nobel laureate Richard Feynman outlined in his 1959 lecture, "There's plenty of room at the bottom", the enormous possibility of producing and visualising things at smaller scales. The advent of advanced scanning and transmission electron microscopy and high-resolution microscopy has begun to open the door to visualise host-pathogen interactions at smaller scales, and spinning disc confocal and two-photon microscopy has improved our ability to study these events in real time in three dimensions. The aim of this review is to illustrate some of the advances in understanding host-fungal interactions that have been made in recent years in particular those relating to the interactions of live fungal pathogens with phagocytes. Dynamic imaging of host-pathogen interactions has recently revealed novel detail and unsuspected mechanistic insights, facilitating the dissection of the phagocytic process into its component parts. Here, we will highlight advances in our knowledge of host-fungal pathogen interactions, including the specific effects of fungal cell viability, cell wall composition and morphogenesis on the phagocytic process and try to define the relative contributions of neutrophils and macrophages to the clearance of fungal pathogens in vitro and the infected host.

Unlabelled: Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation., Importance: Innate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand how C. albicans viability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host., (Copyright © 2014 Bain et al.)

Metabolism is integral to the pathogenicity of Candida albicans, a major fungal pathogen of humans. As well as providing the platform for nutrient assimilation and growth in diverse host niches, metabolic adaptation affects the susceptibility of C. albicans to host-imposed stresses and antifungal drugs, the expression of key virulence factors, and fungal vulnerability to innate immune defences. These effects, which are driven by complex regulatory networks linking metabolism, morphogenesis, stress adaptation, and cell wall remodelling, influence commensalism and infection. Therefore, current concepts of Candida-host interactions must be extended to include the impact of metabolic adaptation upon pathogenicity and immunogenicity., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress--a phenomenon we term stress pathway interference--lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy. Importance: The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance., (Copyright © 2014 Kaloriti et al.)

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90-100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3p-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected.

The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus.

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.

Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology., (© 2014 The Authors. European Journal of Immunology published by Wiley-VCH Verlag GmbH & Co. KGaA Weinheim.)

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed "trained immunity." In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2(-/-)) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2(-/-) mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2(-/-) macrophages and neutrophils. Neutrophils of Dectin-2(-/-) mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip., (Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The soil-borne plant pathogen Fusarium oxysporum causes life-threatening invasive fusariosis in immunocompromised individuals. The mechanism of infection in mammalian hosts is largely unknown. In the present study we show that the symptoms of disseminated fusariosis caused by F. oxysporum in immunosuppressed mice are remarkably similar to those reported in humans. Distinct fungal structures were observed inside the host, depending on the infected organ. Invasive hyphae developed in the heart and kidney, causing massive colonization of the organs. By contrast, chlamydospore-like survival structures were found in lung, spleen and liver. Systemically infected mice also developed skin and eye infections, as well as thrombosis and necrosis in the tail. We further show that F. oxysporum can disseminate and persist in the organs of immunocompetent animals, and that these latent infections can lead to lethal systemic fusariosis if the host is later subjected to immunosuppressive treatment.

Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca(2+) influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca(2+) influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca(2+). Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca(2+). Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca(2+). The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca(2+) influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca(2+)-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post-translational modifications being the addition of O- and N-linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O- and N-linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host-fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease., (© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Unlabelled: An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection., Importance: Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

Background: The Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation., Findings: We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model., Conclusion: Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence.