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1.  A Biphasic Innate Immune MAPK Response Discriminates between the Yeast and Hyphal Forms of Candida albicans in Epithelial Cells 
Cell Host & Microbe  2010;8(3):225-235.
Discriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-κB and a biphasic MAPK response. Activation of NF-κB and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a “danger response” pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.
► NF-κB and MAPK control epithelial effector responses against Candida albicans ► c-Jun activation is independent of morphology and due to fungal cell wall recognition ► MAPK/MKP-1/c-Fos pathway activation is dependent on fungal hyphae and burdens ► MAPK discriminatory response may dictate C. albicans mucosal colonization in vivo
PMCID: PMC2991069  PMID: 20833374
2.  Candida albicans Yeast and Hyphae are Discriminated by MAPK Signaling in Vaginal Epithelial Cells 
PLoS ONE  2011;6(11):e26580.
We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-κB and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The ‘second’ or ‘late’ MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-κB activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
PMCID: PMC3210759  PMID: 22087232
3.  Candida albicans Cell Wall Glycosylation May Be Indirectly Required for Activation of Epithelial Cell Proinflammatory Responses ▿  
Infection and Immunity  2011;79(12):4902-4911.
Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan and N- and O-mannan mutants, we found that N-mannosylation (via Δoch1 and Δpmr1 mutants) and O-mannosylation (via Δpmt1 and Δmnt1 Δmnt2 mutants), but not phosphomannan (via a Δmnn4 mutant) or β-1,2 mannosylation (via Δbmt1 to Δbmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of the N-linked outer chain (corresponding to a Δmnn9 mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1 mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
PMCID: PMC3232641  PMID: 21930756
4.  Activation of MAPK/c-Fos induced responses in oral epithelial cells is specific to Candida albicans and Candida dubliniensis hyphae 
Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-κB and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-κB signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1α) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1α and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.
PMCID: PMC3257392  PMID: 21706283
Candida albicans; Candida dubliniensis; Hypha formation; MAPK; MKP1; c-Fos; NF-κB; Oral epithelium; Innate immunity
5.  Evaluation of the Role of Candida albicans Agglutinin-Like Sequence (Als) Proteins in Human Oral Epithelial Cell Interactions 
PLoS ONE  2012;7(3):e33362.
The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans Δals mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight Δals mutants tested, only the Δals3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the Δals3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
PMCID: PMC3299778  PMID: 22428031
6.  Integrin αXβ2 Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections 
The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in αXβ2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of αXβ2-deficient mice within 9 d. Organs taken from αXβ2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use αXβ2 to control fungal invasion.
PMCID: PMC3424400  PMID: 22844116
7.  Acetylation of MKP-1 and the Control of Inflammation 
Science signaling  2008;1(41):pe44.
Innate immune responses mediated by Toll-like receptors (TLRs), a class of pattern-recognition receptors, play a critical role in the defense against microbial pathogens. However, excessive TLR-mediated responses result in sepsis, autoimmunity, and chronic inflammation. To prevent deleterious activation of TLRs, cells have evolved multiple mechanisms that inhibit innate immune reactions. Stimulation of TLRs induces the expression of the gene encoding the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), a nuclear-localized dual-specificity phosphatase that preferentially dephosphorylates p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in the attenuation of TLR-triggered production of proinflammatory cytokines. MKP-1 is posttranslationally modified by multiple mechanisms, including phosphorylation. A study now demonstrates that MKP-1 is also acetylated on a key lysine residue following stimulation of TLRs. Acetylation of MKP-1 promotes the interaction of MKP-1 with its substrate p38 MAPK, which results in dephosphorylation of p38 MAPK and the inhibition of innate immunity.
PMCID: PMC2613485  PMID: 18922786
8.  The Pathogen Candida albicans Hijacks Pyroptosis for Escape from Macrophages 
mBio  2014;5(2):e00003-14.
The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.
Upon phagocytosis by macrophages, Candida albicans can transition to the hyphal form, which causes macrophage death and enables fungal escape. The current model is that the highly polarized growth of hyphae results in macrophage piercing. This model is challenged by recent reports of C. albicans mutants that form hyphae of wild-type morphology but are defective in killing macrophages. We show that C. albicans causes macrophage cell death by at least two mechanisms. Phase 1 killing (first 6 to 8 h) depends on the activation of the pyroptotic programmed host cell death by fungal hyphae. Phase 2 (up to 24 h) is rapid and depends on robust hyphal formation but is independent of pyroptosis. Our data provide a new model for how the interplay between fungal morphogenesis and activation of a host cell death pathway mediates macrophage killing by C. albicans hyphae.
PMCID: PMC3977349  PMID: 24667705
The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing HEK293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing three-fold in αXβ2 deficient mice compared to wild-type mice. After challenging intravenously with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 days compared to 100% mortality of αXβ2-deficient mice within 9 days. Organs taken from αXβ2-deficient mice 16 hrs after infection revealed a 10-fold increase in fungal invasion into brain and 2-fold increase into liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in liver, Kupffer cells, while in brain, microglial cells employ αXβ2 to control fungal invasion.
PMCID: PMC3424400  PMID: 22844116
integrin αXβ2; Pra1; C. albicans; systemic fungal infections; protection; murine model; adhesion molecules; macrophages; microglia; Kupffer cells
10.  A novel renal epithelial cell in vitro assay to assess Candida albicans virulence 
Virulence  2013;5(2):286-296.
Candida albicans, an opportunistic fungal pathogen, can cause severe systemic infections in susceptible patient groups. Systemic candidiasis is mainly studied in the mouse intravenous challenge model, where progressive infection correlates with increased early renal chemokine levels.
To develop a new in vitro assay to assess C. albicans virulence, which reflects the events occurring in the murine infection model, renal M-1 cortical collecting duct epithelial cells were evaluated as the early producers of cytokines in response to C. albicans. We show that renal epithelial cells respond only to live C. albicans cells capable of forming hyphae, producing chemokines KC and MIP-2, with levels correlating with epithelial cell damage. By assaying epithelial cell responses to strains of known virulence in the murine intravenous challenge model we demonstrate that renal epithelial cells can discriminate between virulent and attenuated strains. This simple, novel assay is a useful initial screen for altered virulence of C. albicans mutants or clinical isolates in vitro and provides an alternative to the mouse systemic infection model.
PMCID: PMC3956504  PMID: 24225657
Candida albicans; virulence; innate immunity; renal epithelium; infection model
11.  The dectin-1/inflammasome pathway is responsible for the induction of protective T-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans 
Journal of Leukocyte Biology  2011;90(2):357-366.
Invading C. albicans hyphae are recognized by macrophages, activate the caspase-1/IL-1β pathway, and lead to the activation of IL-17 pathway to control the C. albicans infection.
In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicans hyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicans hyphae and the subsequent release of active IL-1β in macrophages. Inflammasome activation in macrophages results from differences in cell-wall architecture between yeasts and hyphae and is partly mediated by the dectin-1/Syk pathway. These results define the dectin-1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.
PMCID: PMC3513931  PMID: 21531876
Candida; colonization; invasion; IL-1β; IL-17
12.  Mitogen-activated Protein Kinase Phosphatase-1 Represses c-Jun NH2-terminal Kinase-mediated Apoptosis via NF-κB Regulation* 
The Journal of Biological Chemistry  2008;283(30):21011-21023.
The mechanism regulating radiation-induced anti-apoptotic response, a limiting factor in improving cell radiosensitivity, remains elusive. Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 is the major member of MKPs that dephosphorylates and inactivates MAPK. Here we provide the evidence that MKP-1 was negatively bridging between NF-κB-mediated prosurvival pathway and c-Jun N-terminal kinase (JNK)-mediated proapoptotic response. MKP-1 was induced by γ-radiation and repressed radiation-induced pro-apoptotic status. NF-κB RelA/p50 heterodimer was recruited to MKP-1 gene promoter to induce MKP-1 transcription. Deletion of the NF-κB-binding site or inactivation of NF-κB by its small interfering RNA significantly decreased the radiation-induced MKP-1 promoter activity. In addition, MKP-1-deficient mouse embryonic fibroblasts exhibited a prolonged activation of JNK but not p38 or extracellular signal-regulated kinase subfamilies of MAPKs. The prolonged activation of JNK was not induced by treatment with tumor necrosis factor α or interleukin-6, and inactivation of JNK but not p38 or ERK abolished radiation-induced proapoptotic status, indicating that JNK is specifically inhibited by radiation-induced MKP-1. Three MKP-1 wild type human tumor cell lines treated with MKP-1 small interfering RNA showed an increased proapoptotic response that can be rescued by overexpression of wild type mouse MKP-1. Together, these results suggest that MKP-1 is a NF-κB-mediated prosurvival effector in attenuating JNK-mediated pro-apoptotic response; NF-κB/MKP-1-mediated negative JNK regulation represents a potential therapeutic target for adjusting cell radiosensitivity.
PMCID: PMC2475689  PMID: 18508759
13.  Transcriptional Response of Candida albicans upon Internalization by Macrophages 
Eukaryotic Cell  2004;3(5):1076-1087.
The opportunistic fungal pathogen Candida albicans is both a benign gut commensal and a frequently fatal systemic pathogen. The interaction of C. albicans with the host's innate immune system is the primary factor in this balance; defects in innate immunity predispose the patient to disseminated candidiasis. Because of the central importance of phagocytic cells in defense against fungal infections, we have investigated the response of C. albicans to phagocytosis by mammalian macrophages using genomic transcript profiling. This analysis reveals a dramatic reprogramming of transcription in C. albicans that occurs in two successive steps. In the early phase cells shift to a starvation mode, including gluconeogenic growth, activation of fatty acid degradation, and downregulation of translation. In a later phase, as hyphal growth enables C. albicans to escape from the macrophage, cells quickly resume glycolytic growth. In addition, there is a substantial nonmetabolic response imbedded in the early phase, including machinery for DNA damage repair, oxidative stress responses, peptide uptake systems, and arginine biosynthesis. Further, a surprising percentage of the genes that respond specifically to macrophage contact have no known homologs, suggesting that the organism has undergone substantial evolutionary adaptations to the commensal or pathogen lifestyle. This transcriptional reprogramming is almost wholly absent in the related, but nonpathogenic, yeast Saccharomyces cerevisiae, suggesting that these large-scale and coordinated changes contribute significantly to the ability of this organism to survive and cause disease in vivo.
PMCID: PMC522606  PMID: 15470236
14.  The Role of the IL-12 Cytokine Family in Directing T-Cell Responses in Oral Candidosis 
Candida albicans is an opportunistic fungal pathogen that normally exists as a harmless commensal in humans. In instances where host debilitation occurs, Candida can cause a range of clinical infections, and whilst these are primarily superficial, effecting mucosal membranes, systemic infections can develop in severely immunocompromised individuals. The mechanism of host immunity during commensal carriage of C. albicans has been intensively studied. In this paper, we present the most recent information concerning host recognition of C. albicans leading to cytokine production and the subsequent T-cell responses generated in response to C. albicans. Particular focus is given to the role of the IL-12 cytokine family including IL-12, IL-23, IL-27, and IL-35, in host immunity to Candida. CD4+ T-cells are considered crucial in the regulation of immunity and inflammation. In this regard, the role of Th1/2, helper cells, together with the recently identified Th17 and Treg cells in candidosis will be discussed. Understanding the detailed mechanisms that underlie host immunity to Candida not only will be of benefit in terms of the infections caused by this organism but could also be exploited in the development of therapeutic interventions for other diseases.
PMCID: PMC2963117  PMID: 20981280
15.  Dual-specificity MAP kinase phosphatases (MKPs) 
The Febs Journal  2013;280(2):489-504.
Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative regulatory network that acts to shape the duration, magnitude and spatiotemporal profile of MAP kinase activities in response to both physiological and pathological stimuli. Individual MKPs may exhibit either exquisite specificity towards a single mitogen-activated protein kinase (MAPK) isoform or be able to regulate multiple MAPK pathways in a single cell or tissue. They can act as negative feedback regulators of MAPK activity, but can also provide mechanisms of crosstalk between distinct MAPK pathways and between MAPK signalling and other intracellular signalling modules. In this review, we explore the current state of knowledge with respect to the regulation of MKP expression levels and activities, the mechanisms by which individual MKPs recognize and interact with different MAPK isoforms and their role in the spatiotemporal regulation of MAPK signalling.
PMCID: PMC3594966  PMID: 22812510
dual specificity protein phosphatase; DUSP; ERK; JNK mitogen activated protein kinase; MAPK; MAPK localisation; MAPK phosphatase; MKP; p38
16.  Cytokine and Chemokine Production by Human Oral and Vaginal Epithelial Cells in Response to Candida albicans  
Infection and Immunity  2002;70(2):577-583.
Oropharyngeal and vaginal candidiases are the most common forms of mucosal fungal infections and are primarily caused by Candida albicans, a dimorphic fungal commensal organism of the gastrointestinal and lower female reproductive tracts. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral and vaginal mucosa during C. albicans infections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and since C. albicans is closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines and/or chemokines produced by primary oral and vaginal epithelial cells and cell lines in response to C. albicans. The results showed that proinflammatory cytokines were produced by oral and/or vaginal epithelial cells at various levels constitutively with considerable interleukin-1α (IL-1α) and tumor necrosis factor alpha, but not IL-6, produced in response to C. albicans. In contrast, Th1-type (IL-12 and gamma interferon) and Th2-type-immunoregulatory (IL-10 and transforming growth factor β) cytokines and the chemokines monocyte chemoattractant protein 1 and IL-8 were produced in low to undetectable concentrations with little additional production in response to C. albicans. Taken together, these results indicate that cytokines and chemokines are variably produced by oral and vaginal epithelial cells constitutively, as well as in response to C. albicans, and are predominated by proinflammatory cytokines.
PMCID: PMC127706  PMID: 11796585
17.  The cellular roles of Ccr4-NOT in model and pathogenic fungi—implications for fungal virulence 
Frontiers in Genetics  2013;4:302.
The fungal Ccr4-NOT complex has been implicated in orchestrating gene expression networks that impact on pathways key for virulence in pathogenic species. The activity of Ccr4-NOT regulates cell wall integrity, antifungal drug susceptibility, adaptation to host temperature, and the developmental switches that enable the formation of pathogenic structures, such as filamentous hyphae. Moreover, Ccr4-NOT impacts on DNA repair pathways and genome stability, opening the possibility that this gene regulator could control adaptive responses in pathogens that are driven by chromosomal alterations. Here we provide a synthesis of the cellular roles of the fungal Ccr4-NOT, focusing on pathways important for virulence toward animals. Our review is based on studies in models yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and two species that cause serious human infections, Candida albicans and Cryptococcus neoformans. We hypothesize that the activity of Ccr4-NOT could be targeted for future antifungal drug discovery, a proposition supported by the fact that inactivation of the genes encoding subunits of Ccr4-NOT in C. albicans and C. neoformans reduces virulence in the mouse infection model. We performed bioinformatics analysis to identify similarities and differences between Ccr4-NOT subunits in fungi and animals, and discuss this knowledge in the context of future antifungal strategies.
PMCID: PMC3868889  PMID: 24391665
Ccr4-NOT; fungal pathogen; cell wall; stress adaptation; Candida albicans; Cryptococcus neoformans
18.  Mitogen-activated protein kinase (MKP)-1 in immunology, physiology, and disease 
Life sciences  2011;90(7-8):237-248.
Mitogen-activated protein kinases (MAPKs) are key regulators of cellular physiology and immune responses and abnormality in MAPKs is implicated in many diseases. MAPKs are activated by MAPK kinases through phosphorylation of the threonine and tyrosine residues in the conserved Thr-Xaa-Tyr domain, where Xaa represents amino acid residues characteristic of distinct MAPK subfamilies. Since MAPKs play a crucial role in a variety of cellular processes, a delicate regulatory network has evolved to control their activities. Over the past two decades, a group of dual specificity MAPK phosphatases (MKPs) have been identified that deactivate MAPKs. Since MAPKs can enhance MKP activities, MKPs are considered as an important feedback control mechanism that limits the MAPK cascades. This review outlines the role of MKP-1, a prototypical MKP family member, in physiology and disease. We will first discuss the basic biochemistry and regulation of MKP-1. Next, we will present the current consensus on the immunological and physiological functions of MKP-1 in infectious, inflammatory, metabolic, and nervous system diseases as revealed by studies using animal models. We will also discuss the emerging evidence implicating MKP-1 in human disorders. Finally, we will conclude with a discussion of the potential for pharmacomodulation of MKP-1 expression.
PMCID: PMC3465723  PMID: 22197448
MKP-1; MAPK; immunology; mouse; human; inflammation
19.  Candida albicans Modulates Host Defense by Biosynthesizing the Pro-Resolving Mediator Resolvin E1 
PLoS ONE  2007;2(12):e1316.
Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.
PMCID: PMC2134765  PMID: 18091990
20.  Assessing the advantage of morphological changes in Candida albicans: a game theoretical study 
A range of attributes determines the virulence of human pathogens. During interactions with their hosts, pathogenic microbes often undergo transitions between distinct stages, and the ability to switch between these can be directly related to the disease process. Understanding the mechanisms and dynamics of these transitions is a key factor in understanding and combating infectious diseases. The human fungal pathogen Candida albicans exhibits different morphotypes at different stages during the course of infection (candidiasis). For example, hyphae are considered to be the invasive form, which causes tissue damage, while yeast cells are predominant in the commensal stage. Here, we described interactions of C. albicans with its human host in a game theoretic model. In the game, players are fungal cells. Each fungal cell can adopt one of the two strategies: to exist as a yeast or hyphal cell. We characterized the ranges of model parameters in which the coexistence of both yeast and hyphal forms is plausible. Stability analysis of the system showed that, in theory, a reduced ability of the host to specifically recognize yeast and hyphal cells can result in bi-stability of the microbial populations' profile. Inspired by the model analysis we reasoned that the types of microbial interactions can change during invasive candidiasis. We found that positive cooperation among fungal cells occurs in mild infections and an enhanced tendency to invade the host is associated with negative cooperation. The model can easily be extended to multi-player systems with direct application to identifying individuals that enhance either positive or negative cooperation. Results of the modeling approach have potential application in developing treatment strategies.
PMCID: PMC3915147  PMID: 24567730
Candida albicans; host-pathogen interactions; pair-wise context; mathematical modeling; evolutionary game theory
21.  Candida albicans infection inhibits macrophage cell division and proliferation 
Fungal Genetics and Biology  2012;49(9):679-680.
► First demonstration of post-mitotic fusion of macrophages infected with Candida albicans. ► Post-mitotic fusion was attributed to C. albicans hyphae but not yeast form cells. ► Post-mitotic fusion may inhibit macrophage proliferation and the formation of new uninfected phagocytes.
The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.
PMCID: PMC3430961  PMID: 22634272
Candida albicans; Macrophage; Phagocytosis; Mitosis; Innate immunology
22.  Sequential Activation of ERK and Repression of JNK by Scatter Factor/Hepatocyte Growth Factor in Madin-Darby Canine Kidney Epithelial Cells 
Molecular Biology of the Cell  2000;11(11):3751-3763.
The scattering of Madin-Darby canine kidney (MDCK) epithelial cells by scatter factor/hepatocyte growth factor (SF/HGF) is associated with transcriptional induction of the urokinase gene, which occurs essentially through activation of an EBS/AP1 response element. We have investigated the signal transduction pathways leading to this transcriptional response. We found that SF/HGF induces rapid and sustained phosphorylation of the extracellular signal-regulated kinase (ERK) MAPK while stimulating weakly and then repressing phosphorylation of the JUN N-terminal kinase (JNK) MAPK for several hours. This delayed repression of JNK was preceded by phosphorylation of the MKP2 phosphatase, and both MKP2 induction and JNK dephosphorylation were under the control of MEK, the upstream kinase of ERK. ERK and MKP2 stimulate the EBS/AP1-dependent transcriptional response to SF/HGF, but not JNK, which inhibits this response. We further demonstrated that depending on cell density, the RAS-ERK-MKP2 pathway controls this transrepressing effect of JNK. Together, these data demonstrate that in a sequential manner SF/HGF activates ERK and MKP2, which in turn dephosphorylates JNK. This sequence of events provides a model for efficient cell scattering by SF/HGF at low cell density.
PMCID: PMC15034  PMID: 11071904
23.  CAP1, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of Candida albicans 
Journal of Bacteriology  2001;183(10):3211-3223.
In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.
PMCID: PMC95223  PMID: 11325951
24.  Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis 
PLoS ONE  2012;7(11):e46020.
Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.
PMCID: PMC3491010  PMID: 23139737
25.  The HOG Pathway Is Critical for the Colonization of the Mouse Gastrointestinal Tract by Candida albicans 
PLoS ONE  2014;9(1):e87128.
The opportunistic pathogen Candida albicans is a frequent inhabitant of the human gastrointestinal tract where it usually behaves as a harmless commensal. In this particular niche, it needs to adapt to the different micro environments that challenge its survival within the host. In order to determine those factors involved in gut adaptation, we have used a gastrointestinal model of colonization in mouse to trace the behaviour of fungal cells. We have developed a genetic labelling system based on the complementary spectral properties of the fluorescent proteins GFP and a new C. albicans codon-adapted RFP (dTOM2) that allow a precise quantification of the fungal population in the gut via standard in vitro cultures or flow cytometry. This methodology has allowed us to determine the role of the three MAP kinase pathways of C. albicans (mediated by the MAPK Mkc1, Cek1 or Hog1) in mouse gut colonization via competitive assays with MAPK pathway mutants and their isogenic wild type strain. This approach reveals the signalling through HOG pathway as a critical factor influencing the establishment of C. albicans in the mouse gut. Less pronounced effects for mkc1 or cek1 mutants were found, only evident after 2–3 weeks of colonization. We have also seen that hog1 mutants is defective in adhesion to the gut mucosa and sensitive to bile salts. Finally, we have developed a genetic strategy for the in vivo excision (tetracycline-dependent) of any specific gene during the course of colonization in this particular niche, allowing the analysis of its role during gut colonization.
PMCID: PMC3903619  PMID: 24475243

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