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Identification and functional characterization of Rca1, a transcription factor involved in both antifungal susceptibility and host response in Candida albicans.
FungiNet C - Candida projects

Abstract (Expand)

The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida … albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response.

Authors: P. Vandeputte, S. Pradervand, F. Ischer, A. T. Coste, S. Ferrari, K. Harshman, D. Sanglard

Date Published: No date defined

Journal: Eukaryot Cell

Enemies and brothers in arms: Candida albicans and gram-positive bacteria.
FungiNet C - Candida projects

Abstract (Expand)

Candida albicans is an important human opportunistic fungal pathogen which is frequently found as part of the normal human microbiota. It is well accepted that the fungus interacts with other components … of the resident microbiota and that this impacts the commensal or pathogenic outcome of C. albicans colonization. Different types of interactions, including synergism or antagonism, contribute to a complex balance between the multitude of different species. Mixed biofilms of C. albicans and streptococci are a well-studied example of a mutualistic interaction often potentiating the virulence of the individual members. In contrast, other bacteria like lactobacilli are known to antagonize C. albicans, and research has just started elucidating the mechanisms behind these interactions. This scenario is even more complicated by a third player, the host. This review focuses on interactions between C. albicans and gram-positive bacteria whose investigation will without doubt ultimately help understanding C. albicans infections.

Authors: None

Date Published: No date defined

Journal: Cellular Microbiology

Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood.
FungiNet C - Candida projects, C5

Abstract (Expand)

Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida … species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts.

Authors: S. Machata, S. Sreekantapuram, Kerstin Hünniger, Oliver Kurzai, C. Dunker, K. Schubert, Wibke Krüger, B. Schulze-Richter, C. Speth, G. Rambach, Ilse Jacobsen

Date Published: 1st Feb 2021

Journal: Front Immunol

Molecular analyses identifies new domains and structural differences among Streptococcus pneumoniae immune evasion proteins PspC and Hic.
FungiNet C - Candida projects, C6 (E)

Abstract (Expand)

The PspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles, … it was assumed for a long time that these pneumococcal surface proteins represent a protein family comprised of eleven subgroups. Recently, however, the evaluation of more proteins revealed a greater diversity of individual proteins. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are also present in other virulence factors encoded by pneumococci and other bacterial pathogens. This knowledge improved pattern evaluation at the level of full-length proteins, allowed a sequence comparison at the domain level and identified domains with a modular composition. This novel strategy increased understanding of individual proteins variability and modular domain composition, enabled a structural and functional characterization at the domain level and furthermore revealed substantial structural differences between PspC and Hic proteins. Given the exceptional genomic diversity of the multifunctional PspC and Hic proteins a detailed structural and functional evaluation need to be performed at the strain level. Such knowledge will also be useful for molecular strain typing and characterizing PspC and Hic proteins from new clinical S. pneumoniae strains.

Authors: S. Du, C. Vilhena, S. King, A. Sahagun-Ruiz, S. Hammerschmidt, Christine Skerka, Peter Zipfel

Date Published: 18th Jan 2021

Journal: Sci Rep

Active neutrophil responses counteract Candida albicans burn wound infection of ex vivo human skin explants.
FungiNet C - Candida projects, C2

Abstract (Expand)

Burn wounds are highly susceptible sites for colonization and infection by bacteria and fungi. Large wound surface, impaired local immunity, and broad-spectrum antibiotic therapy support growth of … opportunistic fungi such as Candida albicans, which may lead to invasive candidiasis. Currently, it remains unknown whether depressed host defenses or fungal virulence drive the progression of burn wound candidiasis. Here we established an ex vivo burn wound model, where wounds were inflicted by applying preheated soldering iron to human skin explants, resulting in highly reproducible deep second-degree burn wounds. Eschar removal by debridement allowed for deeper C. albicans penetration into the burned tissue associated with prominent filamentation. Active migration of resident tissue neutrophils towards the damaged tissue and release of pro-inflammatory cytokine IL-1beta accompanied the burn. The neutrophil recruitment was further increased upon supplementation of the model with fresh immune cells. Wound area and depth decreased over time, indicating healing of the damaged tissue. Importantly, prominent neutrophil presence at the infected site correlated to the limited penetration of C. albicans into the burned tissue. Altogether, we established a reproducible burn wound model of candidiasis using ex vivo human skin explants, where immune responses actively control the progression of infection and promote tissue healing.

Authors: C. von Muller, F. Bulman, L. Wagner, D. Rosenberger, A. Marolda, Oliver Kurzai, P. Eissmann, Ilse Jacobsen, B. Perner, P. Hemmerich, Slavena Vylkova

Date Published: 11th Dec 2020

Journal: Sci Rep

The impact of the Fungus-Host-Microbiota interplay upon Candida albicans infections: current knowledge and new perspectives.
FungiNet C - Candida projects, C5

Abstract (Expand)

Candida albicans is a major fungal pathogen of humans. It exists as a commensal in the oral cavity, gut or genital tract of most individuals, constrained by the local microbiota, epithelial barriers … and immune defences. Their perturbation can lead to fungal outgrowth and the development of mucosal infections such as oropharyngeal or vulvovaginal candidiasis, and patients with compromised immunity are susceptible to life-threatening systemic infections. The importance of the interplay between fungus, host and microbiota in driving the transition from C. albicans commensalism to pathogenicity is widely appreciated. However, the complexity of these interactions, and the significant impact of fungal, host and microbiota variability upon disease severity and outcome, are less well understood. Therefore, we summarise the features of the fungus that promote infection, and how genetic variation between clinical isolates influences pathogenicity. We discuss antifungal immunity, how this differs between mucosae, and how individual variation influences a person's susceptibility to infection. Also, we describe factors that influence the composition of gut, oral and vaginal microbiotas, and how these affect fungal colonisation and antifungal immunity. We argue that a detailed understanding of these variables, which underlie fungal-host-microbiota interactions, will present opportunities for directed antifungal therapies that benefit vulnerable patients.

Authors: C. d'Enfert, A. K. Kaune, L. R. Alaban, S. Chakraborty, N. Cole, M. Delavy, D. Kosmala, B. Marsaux, R. Frois-Martins, M. Morelli, D. Rosati, M. Valentine, Z. Xie, Y. Emritloll, P. A. Warn, F. Bequet, M. E. Bougnoux, S. Bornes, Mark Gresnigt, Bernhard Hube, Ilse Jacobsen, M. Legrand, S. Leibundgut-Landmann, C. Manichanh, C. A. Munro, M. G. Netea, K. Queiroz, K. Roget, V. Thomas, C. Thoral, P. Van den Abbeele, A. W. Walker, A. J. P. Brown

Date Published: 24th Nov 2020

Journal: FEMS Microbiol Rev

Quantification of Factor H Mediated Self vs. Non-self Discrimination by Mathematical Modeling.
FungiNet C - Candida projects, B4

Abstract (Expand)

The complement system is part of the innate immune system and plays an important role in the host defense against infectious pathogens. One of the main effects is the opsonization of foreign invaders … and subsequent uptake by phagocytosis. Due to the continuous default basal level of active complement molecules, a tight regulation is required to protect the body's own cells (self cells) from opsonization and from complement damage. A major complement regulator is Factor H, which is recruited from the fluid phase and attaches to cell surfaces where it effectively controls complement activation. Besides self cells, pathogens also have the ability to bind Factor H; they can thus escape opsonization and phagocytosis causing severe infections. In order to advance our understanding of the opsonization process at a quantitative level, we developed a mathematical model for the dynamics of the complement system-termed DynaCoSys model-that is based on ordinary differential equations for cell surface-bound molecules and on partial differential equations for concentration profiles of the fluid phase molecules in the environment of cells. This hybrid differential equation approach allows to model the complement cascade focusing on the role of active C3b in the fluid phase and on the cell surface as well as on its inactivation on the cell surface. The DynaCoSys model enables us to quantitatively predict the conditions under which Factor H mediated complement evasion occurs. Furthermore, investigating the quantitative impact of model parameters by a sensitivity analysis, we identify the driving processes of complement activation and regulation in both the self and non-self regime. The two regimes are defined by a critical Factor H concentration on the cell surface and we use the model to investigate the differential impact of complement model parameters on this threshold value. The dynamic modeling on the surface of pathogens are further relevant to understand pathophysiological situations where Factor H mutants and defective Factor H binding to target surfaces results in pathophysiology such as renal and retinal disease. In the future, this DynaCoSys model will be extended to also enable evaluating treatment strategies of complement-related diseases.

Authors: A. Tille, Teresa Lehnert, Peter Zipfel, Marc Thilo Figge

Date Published: 5th Oct 2020

Journal: Front Immunol

Salt generates antiinflammatory Th17 cells but amplifies pathogenicity in proinflammatory cytokine microenvironments.
FungiNet C - Candida projects, C5

Abstract (Expand)

Th cells integrate signals from their microenvironment to acquire distinct specialization programs for efficient clearance of diverse pathogens or for immunotolerance. Ionic signals have recently been … demonstrated to affect T cell polarization and function. Sodium chloride (NaCl) was proposed to accumulate in peripheral tissues upon dietary intake and to promote autoimmunity via the Th17 cell axis. Here, we demonstrate that high-NaCl conditions induced a stable, pathogen-specific, antiinflammatory Th17 cell fate in human T cells in vitro. The p38/MAPK pathway, involving NFAT5 and SGK1, regulated FoxP3 and IL-17A expression in high-NaCl conditions. The NaCl-induced acquisition of an antiinflammatory Th17 cell fate was confirmed in vivo in an experimental autoimmune encephalomyelitis (EAE) mouse model, which demonstrated strongly reduced disease symptoms upon transfer of T cells polarized in high-NaCl conditions. However, NaCl was coopted to promote murine and human Th17 cell pathogenicity, if T cell stimulation occurred in a proinflammatory and TGF-beta-low cytokine microenvironment. Taken together, our findings reveal a context-dependent, dichotomous role for NaCl in shaping Th17 cell pathogenicity. NaCl might therefore prove beneficial for the treatment of chronic inflammatory diseases in combination with cytokine-blocking drugs.

Authors: J. Matthias, S. Heink, F. Picard, J. Zeitrag, A. Kolz, Y. Y. Chao, D. Soll, G. P. de Almeida, E. Glasmacher, Ilse Jacobsen, T. Riedel, A. Peters, S. Floess, J. Huehn, D. Baumjohann, M. Huber, T. Korn, C. E. Zielinski

Date Published: 1st Sep 2020

Journal: J Clin Invest

Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions.
FungiNet C - Candida projects

Abstract (Expand)

Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into … focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced beta-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient.IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

Authors: F. Gerwien, C. Dunker, Philipp Brandt, E. Garbe, Ilse Jacobsen, Slavena Vylkova

Date Published: 19th Aug 2020

Journal: mSphere

The Transcription Factor Stp2 Is Important for Candida albicans Biofilm Establishment and Sustainability.
FungiNet C - Candida projects, B4

Abstract (Expand)

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. … The sensing and metabolism of amino acids play important roles during various phases of biofilm development - from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2Delta deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2Delta biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4, the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms.

Authors: B. Bottcher, B. Hoffmann, E. Garbe, T. Weise, Z. Cseresnyes, Philipp Brandt, Stefanie Dietrich, D. Driesch, Marc Thilo Figge, Slavena Vylkova

Date Published: 20th May 2020

Journal: Front Microbiol

Enolase From Aspergillus fumigatus Is a Moonlighting Protein That Binds the Human Plasma Complement Proteins Factor H, FHL-1, C4BP, and Plasminogen.
FungiNet A - Aspergillus projects, FungiNet C - Candida projects, B4

Abstract (Expand)

The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting … immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.

Authors: Prasad Dasari, Naile Koleci, Iordana Shopova, D. Wartenberg, Niklas Beyersdorf, Stefanie Dietrich, A. Sahagun-Ruiz, Marc Thilo Figge, Christine Skerka, Axel Brakhage, Peter Zipfel

Date Published: 12th Dec 2019

Journal: Front Immunol

Regulation of the Germinal Center Reaction and Somatic Hypermutation Dynamics by Homologous Recombination.
FungiNet C - Candida projects, B4

Abstract (Expand)

During somatic hypermutation (SHM) of Ig genes in germinal center B cells, lesions introduced by activation-induced cytidine deaminase are processed by multiple error-prone repair pathways. Although … error-free repair by homologous recombination (HR) is crucial to prevent excessive DNA strand breakage at activation-induced cytidine deaminase off-target genes, its role at the hypermutating Ig locus in the germinal center is unexplored. Using B cell-specific inactivation of the critical HR factor Brca2, we detected decreased proliferation, survival, and thereby class switching of ex vivo-activated B cells. Intriguingly, an HR defect allowed for a germinal center reaction and affinity maturation in vivo, albeit at reduced amounts. Analysis of SHM revealed that a certain fraction of DNA lesions at C:G bp was indeed repaired in an error-free manner via Brca2 instead of being processed by error-prone translesion polymerases. By applying a novel pseudo-time in silico analysis of mutational processes, we found that the activity of A:T mutagenesis during SHM increased during a germinal center reaction, but this was in part defective in Brca2-deficient mice. These mutation pattern changes in Brca2-deficient B cells were mostly specific for the Ig V region, suggesting a local or time-dependent need for recombination repair to survive high rates of SHM and especially A:T mutagenesis.

Authors: G. Hirth, Carl-Magnus Svensson, K. Bottcher, S. Ullrich, Marc Thilo Figge, Berit Jungnickel

Date Published: 15th Sep 2019

Journal: J Immunol

A three-dimensional immunocompetent intestine-on-chip model as in vitro platform for functional and microbial interaction studies.
FungiNet C - Candida projects, B4

Abstract (Expand)

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, … and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.

Authors: M. Maurer, M. S. Gresnigt, A. Last, T. Wollny, F. Berlinghof, R. Pospich, Z. Cseresnyes, A. Medyukhina, K. Graf, M. Groger, M. Raasch, F. Siwczak, S. Nietzsche, Ilse Jacobsen, Marc Thilo Figge, Bernhard Hube, O. Huber, A. S. Mosig

Date Published: 10th Aug 2019

Journal: Biomaterials

Serum FHR1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies.
FungiNet total, FungiNet C - Candida projects, C4 (E)

Abstract (Expand)

Persistent inflammation is a hallmark of many human diseases, including anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis. Here, we describe a dominant trigger of … inflammation: human serum factor H-related protein FHR1. In vitro, this protein selectively binds to necrotic cells via its N-terminus; in addition, it binds near necrotic glomerular sites of AAV patients and necrotic areas in atherosclerotic plaques. FHR1, but not factor H, FHR2 or FHR3 strongly induces inflammasome NLRP3 in blood-derived human monocytes, which subsequently secrete IL-1beta, TNFalpha, IL-18 and IL-6. FHR1 triggers the phospholipase C-pathway via the G-protein coupled receptor EMR2 independent of complement. Moreover, FHR1 concentrations of AAV patients negatively correlate with glomerular filtration rates and associate with the levels of inflammation and progressive disease. These data highlight an unexpected role for FHR1 during sterile inflammation, may explain why FHR1-deficiency protects against certain diseases, and identifies potential targets for treatment of auto-inflammatory diseases.

Authors: Sarah Irmscher, S. R. Brix, Peter Zipfel, Luke Donald Halder, S. Mutluturk, S. Wulf, E. Girdauskas, H. Reichenspurner, R. A. K. Stahl, Berit Jungnickel, T. Wiech, Peter Zipfel, Christine Skerka

Date Published: 4th Jul 2019

Journal: Nat Commun

Automated tracking of label-free cells with enhanced recognition of whole tracks.
FungiNet C - Candida projects, B4

Abstract (Expand)

Migration and interactions of immune cells are routinely studied by time-lapse microscopy of in vitro migration and confrontation assays. To objectively quantify the dynamic behavior of cells, software … tools for automated cell tracking can be applied. However, many existing tracking algorithms recognize only rather short fragments of a whole cell track and rely on cell staining to enhance cell segmentation. While our previously developed segmentation approach enables tracking of label-free cells, it still suffers from frequently recognizing only short track fragments. In this study, we identify sources of track fragmentation and provide solutions to obtain longer cell tracks. This is achieved by improving the detection of low-contrast cells and by optimizing the value of the gap size parameter, which defines the number of missing cell positions between track fragments that is accepted for still connecting them into one track. We find that the enhanced track recognition increases the average length of cell tracks up to 2.2-fold. Recognizing cell tracks as a whole will enable studying and quantifying more complex patterns of cell behavior, e.g. switches in migration mode or dependence of the phagocytosis efficiency on the number and type of preceding interactions. Such quantitative analyses will improve our understanding of how immune cells interact and function in health and disease.

Authors: N. Al-Zaben, A. Medyukhina, Stefanie Dietrich, A. Marolda, Kerstin Hünniger, Oliver Kurzai, Marc Thilo Figge

Date Published: 1st Mar 2019

Journal: Sci Rep

Aspf2 From Aspergillus fumigatus Recruits Human Immune Regulators for Immune Evasion and Cell Damage.
FungiNet A - Aspergillus projects, FungiNet C - Candida projects, B4

Abstract (Expand)

The opportunistic fungal pathogen Aspergillus fumigatus can cause life-threatening infections, particularly in immunocompromised patients. Most pathogenic microbes control host innate immune responses … at the earliest time, already before infiltrating host immune cells arrive at the site of infection. Here, we identify Aspf2 as the first A. fumigatus Factor H-binding protein. Aspf2 recruits several human plasma regulators, Factor H, factor-H-like protein 1 (FHL-1), FHR1, and plasminogen. Factor H contacts Aspf2 via two regions located in SCRs6-7 and SCR20. FHL-1 binds via SCRs6-7, and FHR1 via SCRs3-5. Factor H and FHL-1 attached to Aspf2-maintained cofactor activity and assisted in C3b inactivation. A Deltaaspf2 knockout strain was generated which bound Factor H with 28% and FHL-1 with 42% lower intensity. In agreement with less immune regulator acquisition, when challenged with complement-active normal human serum, Deltaaspf2 conidia had substantially more C3b (>57%) deposited on their surface. Consequently, Deltaaspf2 conidia were more efficiently phagocytosed (>20%) and killed (44%) by human neutrophils as wild-type conidia. Furthermore, Aspf2 recruited human plasminogen and, when activated by tissue-type plasminogen activator, newly generated plasmin cleaved the chromogenic substrate S2251 and degraded fibrinogen. Furthermore, plasmin attached to conidia damaged human lung epithelial cells, induced cell retraction, and caused matrix exposure. Thus, Aspf2 is a central immune evasion protein and plasminogen ligand of A. fumigatus. By blocking host innate immune attack and by disrupting human lung epithelial cell layers, Aspf2 assists in early steps of fungal infection and likely allows tissue penetration.

Authors: Prasad Dasari, Iordana Shopova, M. Stroe, D. Wartenberg, H. Martin-Dahse, Niklas Beyersdorf, P. Hortschansky, Stefanie Dietrich, Z. Cseresnyes, Marc Thilo Figge, M. Westermann, Christine Skerka, Axel Brakhage, Peter Zipfel

Date Published: 1st Sep 2018

Journal: Front Immunol

Molecular signatures of liver dysfunction are distinct in fungal and bacterial infections in mice.
FungiNet C - Candida projects, B4

Abstract (Expand)

Rationale: The liver is a central organ not only for metabolism but also immune function. Life-threatening infections of both bacterial and fungal origin can affect liver function but it is yet unknown … whether molecular changes differ depending on the pathogen. We aimed to determine whether the hepatic host response to bacterial and fungal infections differs in terms of hepatic metabolism and liver function. Methods: We compared murine models of infection, including bacterial peritoneal contamination and infection (PCI), intraperitoneal and systemic C. albicans infection, at 6 and 24 h post-infection, to sham controls. The molecular hepatic host response was investigated by the detection of regulatory modules based on large-scale protein-protein interaction networks and expression data. Topological analysis of these regulatory modules was used to reveal infection-specific biological processes and molecular mechanisms. Intravital microscopy and immunofluorescence microscopy were used to further analyze specific aspects of pathophysiology such as cholestasis. Results: Down-regulation of lipid catabolism and bile acid synthesis was observed after 6 h in all infection groups. Alterations in lipid catabolism were characterized by accumulation of long chain acylcarnitines and defective beta-oxidation, which affected metabolism by 6 h. While PCI led to an accumulation of unconjugated bile acids (BA), C. albicans infection caused accumulation of conjugated BA independent of the route of infection. Hepatic dye clearance and transporter expression revealed reduced hepatic uptake in fungal infections vs. defects in secretion following polybacterial infection. Conclusion: Molecular phenotypes of lipid accumulation and cholestasis allow differentiation between pathogens as well as routes of infection at early stages in mice. Targeted metabolomics could be a useful tool for the profiling of infected/septic patients and the type of pathogen, with subsequent customization and targeting of therapy.

Authors: Barbara Schaarschmidt, S. Vlaic, A. Medyukhina, S. Neugebauer, S. Nietzsche, F. A. Gonnert, J. Rodel, M. Singer, M. Kiehntopf, Marc Thilo Figge, Ilse Jacobsen, Michael Bauer, A. T. Press

Date Published: 8th Aug 2018

Journal: Theranostics

Quantitative Simulations Predict Treatment Strategies Against Fungal Infections in Virtual Neutropenic Patients.
FungiNet C - Candida projects, B4

Abstract (Expand)

The condition of neutropenia, i.e., a reduced absolute neutrophil count in blood, constitutes a major risk factor for severe infections in the affected patients. Candida albicans and Candida glabrata … are opportunistic pathogens and the most prevalent fungal species in the human microbiota. In immunocompromised patients, they can become pathogenic and cause infections with high mortality rates. In this study, we use a previously established approach that combines experiments and computational models to investigate the innate immune response during blood stream infections with the two fungal pathogens C. albicans and C. glabrata. First, we determine immune-reaction rates and migration parameters under healthy conditions. Based on these findings, we simulate virtual patients and investigate the impact of neutropenic conditions on the infection outcome with the respective pathogen. Furthermore, we perform in silico treatments of these virtual patients by simulating a medical treatment that enhances neutrophil activity in terms of phagocytosis and migration. We quantify the infection outcome by comparing the response to the two fungal pathogens relative to non-neutropenic individuals. The analysis reveals that these fungal infections in neutropenic patients can be successfully cleared by cytokine treatment of the remaining neutrophils; and that this treatment is more effective for C. glabrata than for C. albicans.

Authors: Sandra Timme, Teresa Lehnert, M. T. E. Prausse, Kerstin Hünniger, I. Leonhardt, Oliver Kurzai, Marc Thilo Figge

Date Published: 20th Apr 2018

Journal: Front Immunol

Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood.
FungiNet C - Candida projects, B4

Abstract (Expand)

Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune … response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism.

Authors: M. T. E. Prausse, Teresa Lehnert, Sandra Timme, Kerstin Hünniger, I. Leonhardt, Oliver Kurzai, Marc Thilo Figge

Date Published: 6th Apr 2018

Journal: Front Immunol

Streptococcus pneumoniae From Patients With Hemolytic Uremic Syndrome Binds Human Plasminogen via the Surface Protein PspC and Uses Plasmin to Damage Human Endothelial Cells.
FungiNet C - Candida projects, B4

Abstract (Expand)

Pneumococcal hemolytic uremic syndrome (HUS) in children is caused by infections with Streptococcus pneumoniae. Because endothelial cell damage is a hallmark of HUS, we studied how HUS-inducing pneumococci … derived from infant HUS patients during the acute phase disrupt the endothelial layer. HUS pneumococci efficiently bound human plasminogen. These clinical isolates of HUS pneumococci efficiently bound human plasminogen via the bacterial surface proteins Tuf and PspC. When activated to plasmin at the bacterial surface, the active protease degraded fibrinogen and cleaved C3b. Here, we show that PspC is a pneumococcal plasminogen receptor and that plasmin generated on the surface of HUS pneumococci damages endothelial cells, causing endothelial retraction and exposure of the underlying matrix. Thus, HUS pneumococci damage endothelial cells in the blood vessels and disturb local complement homeostasis. Thereby, HUS pneumococci promote a thrombogenic state that drives HUS pathology.

Authors: C. Meinel, G. Sparta, H. M. Dahse, F. Horhold, R. Konig, M. Westermann, S. M. Coldewey, Z. Cseresnyes, Marc Thilo Figge, S. Hammerschmidt, Christine Skerka, Peter Zipfel

Date Published: 17th Jan 2018

Journal: J Infect Dis

Kruppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans
FungiNet A - Aspergillus projects, FungiNet B - Bioinformatics projects, FungiNet C - Candida projects, INF

Abstract (Expand)

Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are … still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Kruppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation.

Authors: K. Czakai, I. Leonhardt, Andreas Dix, M. Bonin, Jörg Linde, Hermann Einsele, Oliver Kurzai, Jürgen Löffler

Date Published: 28th Jun 2016

Journal: Sci Rep

Candida albicans Induces Metabolic Reprogramming in Human NK Cells and Responds to Perforin with a Zinc Depletion Response
FungiNet A - Aspergillus projects, FungiNet C - Candida projects

Abstract (Expand)

As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against … many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin.

Authors: Dragana Slavkovic Lukic, J. Voigt, M. Bouzani, Jürgen Löffler, Daniela Albrecht-Eckardt, Michael Weber, Stefanie Allert, R. Martin, Oliver Kurzai, Kerstin Hünniger

Date Published: 19th May 2016

Journal: Front Microbiol

Candidalysin is a fungal peptide toxin critical for mucosal infection
FungiNet C - Candida projects

Abstract (Expand)

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. … Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Authors: D. L. Moyes, D. Wilson, J. P. Richardson, S. Mogavero, S. X. Tang, J. Wernecke, S. Hofs, R. L. Gratacap, J. Robbins, M. Runglall, C. Murciano, M. Blagojevic, S. Thavaraj, Toni Förster, B. Hebecker, Lydia Kasper, G. Vizcay, S. I. Iancu, N. Kichik, A. Hader, Oliver Kurzai, T. Luo, T. Kruger, O. Kniemeyer, E. Cota, O. Bader, R. T. Wheeler, T. Gutsmann, Bernhard Hube, J. R. Naglik

Date Published: 30th Mar 2016

Journal: Nature

N-Acetylglucosamine-Induced Cell Death in Candida albicans and Its Implications for Adaptive Mechanisms of Nutrient Sensing in Yeasts.
FungiNet total, FungiNet C - Candida projects

Abstract (Expand)

UNLABELLED: Single-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member … of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans' more efficient colonization of the gut. IMPORTANCE: The ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicans uses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicans due to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.

Authors: H. Du, G. Guan, X. Li, M. Gulati, L. Tao, C. Cao, A. D. Johnson, C. J. Nobile, G. Huang

Date Published: 10th Sep 2015

Journal: MBio

Function and Regulation of Cph2 in Candida albicans.
FungiNet C - Candida projects

Abstract (Expand)

Candida albicans is associated with humans as both a harmless commensal organism and a pathogen. Cph2 is a transcription factor whose DNA binding domain is similar to that of mammalian sterol response … element binding proteins (SREBPs). SREBPs are master regulators of cellular cholesterol levels and are highly conserved from fungi to mammals. However, ergosterol biosynthesis is regulated by the zinc finger transcription factor Upc2 in C. albicans and several other yeasts. Cph2 is not necessary for ergosterol biosynthesis but is important for colonization in the murine gastrointestinal (GI) tract. Here we demonstrate that Cph2 is a membrane-associated transcription factor that is processed to release the N-terminal DNA binding domain like SREBPs, but its cleavage is not regulated by cellular levels of ergosterol or oxygen. Chromatin immunoprecipitation sequencing (ChIP-seq) shows that Cph2 binds to the promoters of HMS1 and other components of the regulatory circuit for GI tract colonization. In addition, 50% of Cph2 targets are also bound by Hms1 and other factors of the regulatory circuit. Several common targets function at the head of the glycolysis pathway. Thus, Cph2 is an integral part of the regulatory circuit for GI colonization that regulates glycolytic flux. Transcriptome sequencing (RNA-seq) shows a significant overlap in genes differentially regulated by Cph2 and hypoxia, and Cph2 is important for optimal expression of some hypoxia-responsive genes in glycolysis and the citric acid cycle. We suggest that Cph2 and Upc2 regulate hypoxia-responsive expression in different pathways, consistent with a synthetic lethal defect of the cph2 upc2 double mutant in hypoxia.

Authors: S. Lane, P. Di Lena, K. Tormanen, P. Baldi, H. Liu

Date Published: 6th Sep 2015

Journal: Eukaryot Cell

pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans.
FungiNet C - Candida projects

Abstract (Expand)

As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental conditions. Among them, ambient pH, which changes frequently and affects many biological … processes in this species, is an important factor, and the ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pH promotes white-to-opaque switching under certain culture conditions but represses sexual mating. The Rim101-mediated pH-sensing pathway is involved in the control of pH-regulated white-opaque switching and the mating response. Phr2 and Rim101 could play a major role in acidic pH-induced opaque cell formation. Despite the fact that the cyclic AMP (cAMP) signaling pathway does not play a major role in pH-regulated white-opaque switching and mating, white and opaque cells of the cyr1/cyr1 mutant, which is defective in the production of cAMP, showed distinct growth defects under acidic and alkaline conditions. We further discovered that acidic pH conditions repressed sexual mating due to the failure of activation of the Ste2-mediated alpha-pheromone response pathway in opaque A: cells. The effects of pH changes on phenotypic switching and sexual mating could involve a balance of host adaptation and sexual reproduction in C. albicans.

Authors: Y. Sun, C. Cao, W. Jia, L. Tao, G. Guan, G. Huang

Date Published: 6th Sep 2015

Journal: Eukaryot Cell

Host-pathogen interactions between the human innate immune system and Candida albicans-understanding and modeling defense and evasion strategies
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long … time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.

Authors: Sybille Dühring, S. Germerodt, Christine Skerka, Peter Zipfel, Thomas Dandekar, Stefan Schuster

Date Published: 30th Jun 2015

Journal: Front Microbiol

Bottom-up modeling approach for the quantitative estimation of parameters in pathogen-host interactions
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

Opportunistic fungal pathogens can cause bloodstream infection and severe sepsis upon entering the blood stream of the host. The early immune response in human blood comprises the elimination of pathogens … by antimicrobial peptides and innate immune cells, such as neutrophils or monocytes. Mathematical modeling is a predictive method to examine these complex processes and to quantify the dynamics of pathogen-host interactions. Since model parameters are often not directly accessible from experiment, their estimation is required by calibrating model predictions with experimental data. Depending on the complexity of the mathematical model, parameter estimation can be associated with excessively high computational costs in terms of run time and memory. We apply a strategy for reliable parameter estimation where different modeling approaches with increasing complexity are used that build on one another. This bottom-up modeling approach is applied to an experimental human whole-blood infection assay for Candida albicans. Aiming for the quantification of the relative impact of different routes of the immune response against this human-pathogenic fungus, we start from a non-spatial state-based model (SBM), because this level of model complexity allows estimating a priori unknown transition rates between various system states by the global optimization method simulated annealing. Building on the non-spatial SBM, an agent-based model (ABM) is implemented that incorporates the migration of interacting cells in three-dimensional space. The ABM takes advantage of estimated parameters from the non-spatial SBM, leading to a decreased dimensionality of the parameter space. This space can be scanned using a local optimization approach, i.e., least-squares error estimation based on an adaptive regular grid search, to predict cell migration parameters that are not accessible in experiment. In the future, spatio-temporal simulations of whole-blood samples may enable timely stratification of sepsis patients by distinguishing hyper-inflammatory from paralytic phases in immune dysregulation.

Authors: T. Lehnert, Sandra Timme, J. Pollmacher, Kerstin Hünniger, Oliver Kurzai, Marc Thilo Figge

Date Published: 19th Jun 2015

Journal: Front Microbiol

Neutrophil activation by Candida glabrata but not Candida albicans promotes fungal uptake by monocytes
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, … live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

Authors: S. Duggan, F. Essig, Kerstin Hünniger, Z. Mokhtari, Michael Bauer, T. Lehnert, S. Brandes, A. Hader, Ilse Jacobsen, R. Martin, Marc Thilo Figge, Oliver Kurzai

Date Published: 5th May 2015

Journal: Cell Microbiol

The fungal quorum-sensing molecule farnesol activates innate immune cells but suppresses cellular adaptive immunity
FungiNet C - Candida projects

Abstract (Expand)

Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely … linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and macrophage inflammatory protein 1 alpha [MIP-1alpha]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. IMPORTANCE: Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor.

Authors: I. Leonhardt, S. Spielberg, M. Weber, D. Albrecht-Eckardt, M. Blass, R. Claus, D. Barz, K. Scherlach, C. Hertweck, J. Loffler, Kerstin Hünniger, Oliver Kurzai

Date Published: 19th Mar 2015

Journal: MBio

Host response to Candida albicans bloodstream infection and sepsis
FungiNet C - Candida projects

Abstract (Expand)

Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate … mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management.

Authors: S. Duggan, I. Leonhardt, Kerstin Hünniger, Oliver Kurzai

Date Published: 18th Mar 2015

Journal: Virulence

Biomarker-based classification of bacterial and fungal whole-blood infections in a genome-wide expression study
FungiNet A - Aspergillus projects, FungiNet B - Bioinformatics projects, FungiNet C - Candida projects, INF

Abstract (Expand)

Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used … detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97%) for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83%) for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances.

Authors: Andreas Dix, Kerstin Hünniger, M. Weber, Reinhard Guthke, Oliver Kurzai, Jörg Linde

Date Published: 11th Mar 2015

Journal: Front Microbiol

Candida survival strategies
FungiNet C - Candida projects

Abstract (Expand)

Only few Candida species, e.g., Candida albicans, Candida glabrata, Candida dubliniensis, and Candida parapsilosis, are successful colonizers of a human host. Under certain circumstances these species … can cause infections ranging from superficial to life-threatening disseminated candidiasis. The success of C. albicans, the most prevalent and best studied Candida species, as both commensal and human pathogen depends on its genetic, biochemical, and morphological flexibility which facilitates adaptation to a wide range of host niches. In addition, formation of biofilms provides additional protection from adverse environmental conditions. Furthermore, in many host niches Candida cells coexist with members of the human microbiome. The resulting fungal-bacterial interactions have a major influence on the success of C. albicans as commensal and also influence disease development and outcome. In this chapter, we review the current knowledge of important survival strategies of Candida spp., focusing on fundamental fitness and virulence traits of C. albicans.

Authors: M. Polke, Bernhard Hube, Ilse Jacobsen

Date Published: 24th Feb 2015

Journal: Adv Appl Microbiol

The transcriptional stress response of Candida albicans to weak organic acids.
FungiNet total, FungiNet C - Candida projects

Abstract (Expand)

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of … the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease.

Authors: F. Cottier, A. S. Tan, J. Chen, J. Lum, F. Zolezzi, M. Poidinger, N. Pavelka

Date Published: 1st Feb 2015

Journal: G3 (Bethesda)

Defining the transcriptomic landscape of Candida glabrata by RNA-Seq
FungiNet C - Candida projects, INF

Abstract (Expand)

Candida glabrata is the second most common pathogenic Candida species and has emerged as a leading cause of nosocomial fungal infections. Its reduced susceptibility to antifungal drugs and its close … relationship to Saccharomyces cerevisiae make it an interesting research focus. Although its genome sequence was published in 2004, little is known about its transcriptional dynamics. Here, we provide a detailed RNA-Seq-based analysis of the transcriptomic landscape of C. glabrata in nutrient-rich media, as well as under nitrosative stress and during pH shift. Using RNA-Seq data together with state-of-the-art gene prediction tools, we refined the annotation of the C. glabrata genome and predicted 49 novel protein-coding genes. Of these novel genes, 14 have homologs in S. cerevisiae and six are shared with other Candida species. We experimentally validated four novel protein-coding genes of which two are differentially regulated during pH shift and interaction with human neutrophils, indicating a potential role in host-pathogen interaction. Furthermore, we identified 58 novel non-protein-coding genes, 38 new introns and condition-specific alternative splicing. Finally, our data suggest different patterns of adaptation to pH shift and nitrosative stress in C. glabrata, Candida albicans and S. cerevisiae and thus further underline a distinct evolution of virulence in yeast.

Authors: Jörg Linde, S. Duggan, M. Weber, F. Horn, Patricia Sieber, D. Hellwig, Konstantin Riege, Manja Marz, R. Martin, Reinhard Guthke, Oliver Kurzai

Date Published: 13th Jan 2015

Journal: Nucleic Acids Res

A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a
FungiNet C - Candida projects

Abstract (Expand)

Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are … required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Delta/efg1Delta from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.

Authors: Kerstin Hünniger, K. Bieber, R. Martin, T. Lehnert, Marc Thilo Figge, J. Loffler, R. F. Guo, N. C. Riedemann, Oliver Kurzai

Date Published: 24th Dec 2014

Journal: J Immunol

Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant
FungiNet C - Candida projects, INF

Abstract (Expand)

Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary … adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Delta/efg1Delta mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Delta/efg1Delta strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

Authors: A. Wartenberg, Jörg Linde, R. Martin, M. Schreiner, F. Horn, Ilse Jacobsen, S. Jenull, Thomas Wolf, K. Kuchler, Reinhard Guthke, Oliver Kurzai, A. Forche, C. d'Enfert, S. Brunke, Bernhard Hube

Date Published: 4th Dec 2014

Journal: PLoS Genet

Automated segmentation and tracking of non-rigid objects in time-lapse microscopy videos of polymorphonuclear neutrophils
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

Time-lapse microscopy is an important technique to study the dynamics of various biological processes. The labor-intensive manual analysis of microscopy videos is increasingly replaced by automated … segmentation and tracking methods. These methods are often limited to certain cell morphologies and/or cell stainings. In this paper, we present an automated segmentation and tracking framework that does not have these restrictions. In particular, our framework handles highly variable cell shapes and does not rely on any cell stainings. Our segmentation approach is based on a combination of spatial and temporal image variations to detect moving cells in microscopy videos. This method yields a sensitivity of 99% and a precision of 95% in object detection. The tracking of cells consists of different steps, starting from single-cell tracking based on a nearest-neighbor-approach, detection of cell-cell interactions and splitting of cell clusters, and finally combining tracklets using methods from graph theory. The segmentation and tracking framework was applied to synthetic as well as experimental datasets with varying cell densities implying different numbers of cell-cell interactions. We established a validation framework to measure the performance of our tracking technique. The cell tracking accuracy was found to be >99% for all datasets indicating a high accuracy for connecting the detected cells between different time points.

Authors: S. Brandes, Z. Mokhtari, F. Essig, Kerstin Hünniger, Oliver Kurzai, Marc Thilo Figge

Date Published: 8th Nov 2014

Journal: Med Image Anal

White cells facilitate opposite- and same-sex mating of opaque cells in Candida albicans.
FungiNet C - Candida projects

Abstract (Expand)

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become … mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.

Authors: L. Tao, C. Cao, W. Liang, G. Guan, Q. Zhang, C. J. Nobile, G. Huang

Date Published: 21st Oct 2014

Journal: PLoS Genet

Metabolism in fungal pathogenesis
FungiNet C - Candida projects

Abstract (Expand)

Fungal pathogens must assimilate local nutrients to establish an infection in their mammalian host. We focus on carbon, nitrogen, and micronutrient assimilation mechanisms, discussing how these influence … host-fungus interactions during infection. We highlight several emerging trends based on the available data. First, the perturbation of carbon, nitrogen, or micronutrient assimilation attenuates fungal pathogenicity. Second, the contrasting evolutionary pressures exerted on facultative versus obligatory pathogens have led to contemporary pathogenic fungal species that display differing degrees of metabolic flexibility. The evolutionarily ancient metabolic pathways are conserved in most fungal pathogen, but interesting gaps exist in some species (e.g., Candida glabrata). Third, metabolic flexibility is generally essential for fungal pathogenicity, and in particular, for the adaptation to contrasting host microenvironments such as the gastrointestinal tract, mucosal surfaces, bloodstream, and internal organs. Fourth, this metabolic flexibility relies on complex regulatory networks, some of which are conserved across lineages, whereas others have undergone significant evolutionary rewiring. Fifth, metabolic adaptation affects fungal susceptibility to antifungal drugs and also presents exciting opportunities for the development of novel therapies.

Authors: I. V. Ene, S. Brunke, A. J. Brown, Bernhard Hube

Date Published: 4th Sep 2014

Journal: Cold Spring Harb Perspect Med

Role of Candida albicans Tem1 in mitotic exit and cytokinesis.
FungiNet C - Candida projects

Abstract (Expand)

Candida albicans demonstrates three main growth morphologies: yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely … linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. Here we have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Cells depleted of Tem1 displayed highly polarised growth but ultimately failed to both complete cytokinesis and re-enter the cell cycle following nuclear division. Consistent with its role in activating the mitotic exit network Tem1 localises to spindle pole bodies in a cell cycle-dependent manner. Ultimately, the mitotic exit network in C. albicans appears to co-ordinate the sequential processes of mitotic exit, cytokinesis and cell separation.

Authors: S. W. Milne, J. Cheetham, D. Lloyd, S. Shaw, K. Moore, K. H. Paszkiewicz, S. J. Aves, S. Bates

Date Published: 29th Jun 2014

Journal: Fungal Genet Biol

In vivo imaging of disseminated murine Candida albicans infection reveals unexpected host sites of fungal persistence during antifungal therapy
FungiNet C - Candida projects

Abstract (Expand)

OBJECTIVES: Candida albicans is an important fungal pathogen that can cause life-threatening disseminated infections. To determine the efficacy of therapy in murine models, a determination of renal … fungal burden as cfu is commonly used. However, this approach provides only a snapshot of the current situation in an individual animal and cryptic sites of infection may easily be missed. Thus, we aimed to develop real-time non-invasive imaging to monitor infection in vivo. METHODS: Bioluminescent C. albicans reporter strains were developed based on a bioinformatical approach for codon optimization. The reporter strains were analysed in vitro and in vivo in the murine model of systemic candidiasis. RESULTS: Reporter strains allowed the in vivo monitoring of infection and a determination of fungal burden, with a high correlation between bioluminescence and cfu count. We confirmed the kidney as the main target organ but additionally observed the translocation of C. albicans to the urinary bladder. The treatment of infected mice with caspofungin and fluconazole significantly improved the clinical outcome and clearance of C. albicans from the kidneys; however, unexpectedly, viable fungal cells persisted in the gall bladder. Fungi were secreted with bile and detected in the faeces, implicating the gall bladder as a reservoir for colonization by C. albicans after antifungal therapy. Bile extracts significantly decreased the susceptibility of C. albicans to various antifungals in vitro, thereby probably contributing to its persistence. CONCLUSIONS: Using in vivo imaging, we identified cryptic sites of infection and persistence of C. albicans in the gall bladder during otherwise effective antifungal treatment. Bile appears to directly interfere with antifungal activity.

Authors: Ilse Jacobsen, A. Luttich, Oliver Kurzai, Bernhard Hube, Matthias Brock

Date Published: 20th Jun 2014

Journal: J Antimicrob Chemother

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice
FungiNet C - Candida projects

Abstract (Expand)

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, … as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

Authors: J. Quintin, J. Voigt, R. van der Voort, Ilse Jacobsen, I. Verschueren, Bernhard Hube, E. J. Giamarellos-Bourboulis, J. W. van der Meer, L. A. Joosten, Oliver Kurzai, M. G. Netea

Date Published: 27th May 2014

Journal: Eur J Immunol

Pathogenicity mechanisms and host response during oral Candida albicans infections
FungiNet C - Candida projects

Abstract (Expand)

Oral candidiasis remains one of the most common forms of Candida infections and occurs if the balance between host, Candida and microbiota is disturbed, e.g., by broad spectrum antibiotics or … immunosuppression. In recent years, identification of fungal factors contributing to host cell damage and new insights into host defense mechanisms have significantly extended our understanding of the pathogenesis of oral candidiasis. In this review, we will provide an overview of the pathogenicity mechanisms during oral Candida infections and discuss some approaches by which this knowledge could be transferred into therapeutic approaches.

Authors: B. Hebecker, J. R. Naglik, Bernhard Hube, Ilse Jacobsen

Date Published: 7th May 2014

Journal: Expert Rev Anti Infect Ther

Regulatory networks controlling nitrogen sensing and uptake in Candida albicans
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects, INF

Abstract (Expand)

Nitrogen is one of the key nutrients for microbial growth. During infection, pathogenic fungi like C. albicans need to acquire nitrogen from a broad range of different and changing sources inside the … host. Detecting the available nitrogen sources and adjusting the expression of genes for their uptake and degradation is therefore crucial for survival and growth as well as for establishing an infection. Here, we analyzed the transcriptional response of C. albicans to nitrogen starvation and feeding with the infection-relevant nitrogen sources arginine and bovine serum albumin (BSA), representing amino acids and proteins, respectively. The response to nitrogen starvation was marked by an immediate repression of protein synthesis and an up-regulation of general amino acid permeases, as well as an up-regulation of autophagal processes in its later stages. Feeding with arginine led to a fast reduction in expression of general permeases for amino acids and to resumption of protein synthesis. The response to BSA feeding was generally slower, and was additionally characterized by an up-regulation of oligopeptide transporter genes. From time-series data, we inferred network interaction models for genes relevant in nitrogen detection and uptake. Each individual network was found to be largely specific for the experimental condition (starvation or feeding with arginine or BSA). In addition, we detected several novel connections between regulator and effector genes, with putative roles in nitrogen uptake. We conclude that C. albicans adopts a particular nitrogen response network, defined by sets of specific gene-gene connections for each environmental condition. All together, they form a grid of possible gene regulatory networks, increasing the transcriptional flexibility of C. albicans.

Authors: S. Ramachandra, Jörg Linde, Matthias Brock, Reinhard Guthke, Bernhard Hube, S. Brunke

Date Published: 20th Mar 2014

Journal: PLoS One

Distinct roles of Candida albicans-specific genes in host-pathogen interactions
FungiNet C - Candida projects

Abstract (Expand)

Human fungal pathogens are distributed throughout their kingdom, suggesting that pathogenic potential evolved independently. Candida albicans is the most virulent member of the CUG clade of yeasts and … a common cause of both superficial and invasive infections. We therefore hypothesized that C. albicans possesses distinct pathogenicity mechanisms. In silico genome subtraction and comparative transcriptional analysis identified a total of 65 C. albicans-specific genes (ASGs) expressed during infection. Phenotypic characterization of six ASG-null mutants demonstrated that these genes are dispensable for in vitro growth but play defined roles in host-pathogen interactions. Based on these analyses, we investigated two ASGs in greater detail. An orf19.6688Delta mutant was found to be fully virulent in a mouse model of disseminated candidiasis and to induce higher levels of the proinflammatory cytokine interleukin-1beta (IL-1beta) following incubation with murine macrophages. A pga16Delta mutant, on the other hand, exhibited attenuated virulence. Moreover, we provide evidence that secondary filamentation events (multiple hyphae emerging from a mother cell and hyphal branching) contribute to pathogenicity: PGA16 deletion did not influence primary hypha formation or extension following contact with epithelial cells; however, multiple hyphae and hyphal branching were strongly reduced. Significantly, these hyphae failed to damage host cells as effectively as the multiple hypha structures formed by wild-type C. albicans cells. Together, our data show that species-specific genes of a eukaryotic pathogen can play important roles in pathogenicity.

Authors: D. Wilson, F. L. Mayer, P. Miramon, F. Citiulo, S. Slesiona, Ilse Jacobsen, Bernhard Hube

Date Published: 7th Mar 2014

Journal: Eukaryot Cell

A virtual infection model quantifies innate effector mechanisms and Candida albicans immune escape in human blood
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, … fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost [Formula: see text] of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment-model-experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.

Authors: Kerstin Hünniger, T. Lehnert, K. Bieber, R. Martin, Marc Thilo Figge, Oliver Kurzai

Date Published: 20th Feb 2014

Journal: PLoS Comput Biol

Epithelial invasion outcompetes hypha development during Candida albicans infection as revealed by an image-based systems biology approach
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

Candida albicans is the most common opportunistic fungal pathogen of the human mucosal flora, frequently causing infections. The fungus is responsible for invasive infections in immunocompromised … patients that can lead to sepsis. The yeast to hypha transition and invasion of host-tissue represent major determinants in the switch from benign colonizer to invasive pathogen. A comprehensive understanding of the infection process requires analyses at the quantitative level. Utilizing fluorescence microscopy with differential staining, we obtained images of C. albicans undergoing epithelial invasion during a time course of 6 h. An image-based systems biology approach, combining image analysis and mathematical modeling, was applied to quantify the kinetics of hyphae development, hyphal elongation, and epithelial invasion. The automated image analysis facilitates high-throughput screening and provided quantities that allow for the time-resolved characterization of the morphological and invasive state of fungal cells. The interpretation of these data was supported by two mathematical models, a kinetic growth model and a kinetic transition model, that were developed using differential equations. The kinetic growth model describes the increase in hyphal length and revealed that hyphae undergo mass invasion of epithelial cells following primary hypha formation. We also provide evidence that epithelial cells stimulate the production of secondary hyphae by C. albicans. Based on the kinetic transition model, the route of invasion was quantified in the state space of non-invasive and invasive fungal cells depending on their number of hyphae. This analysis revealed that the initiation of hyphae formation represents an ultimate commitment to invasive growth and suggests that in vivo, the yeast to hypha transition must be under exquisitely tight negative regulation to avoid the transition from commensal to pathogen invading the epithelium.

Authors: F. Mech, D. Wilson, T. Lehnert, Bernhard Hube, M. Thilo Figge

Date Published: 20th Nov 2013

Journal: Cytometry A

Human natural killer cells acting as phagocytes against Candida albicans and mounting an inflammatory response that modulates neutrophil antifungal activity
FungiNet A - Aspergillus projects, FungiNet C - Candida projects

Abstract (Expand)

BACKGROUND: Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activity. Whereas activity of NK cells has been demonstrated against the fungal pathogens Aspergillus fumigatus and … Cryptococcus neoformans, little was known about their interaction with Candida albicans. METHODS: Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry, and enzyme-linked immunosorbent assay. RESULTS: Human NK cells actively recognized C. albicans, resulting in degranulation and secretion of granulocyte-macrophage colony-stimulating factor, interferon gamma, and tumor necrosis factor alpha . Uniquely, activation of NK cells was triggered by actin-dependent phagocytosis. Antifungal activity of NK cells against C. albicans could be detected and mainly attributed to secreted perforin. However, NK cells were unable to inhibit filamentation of C. albicans. Human polymorphonuclear neutrophils (PMNs) counteracted the proinflammatory reaction of NK cells by preventing direct contact between NK cells and the fungal pathogen. Activation of PMNs was enhanced in the presence of NK cells, resulting in increased fungicidal activity. CONCLUSIONS: Our results show a unique pattern of NK cell interaction with C. albicans, which involves direct proinflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cell activation.

Authors: J. Voigt, Kerstin Hünniger, M. Bouzani, Ilse Jacobsen, D. Barz, Bernhard Hube, Jürgen Löffler, Oliver Kurzai

Date Published: 25th Oct 2013

Journal: J Infect Dis

Clotrimazole dampens vaginal inflammation and neutrophil infiltration in response to Candida albicans infection
FungiNet C - Candida projects

Abstract (Expand)

The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the … mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a "danger response" transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.

Authors: D. Wilson, B. Hebecker, D. L. Moyes, P. Miramon, N. Jablonowski, S. Wisgott, S. Allert, J. R. Naglik, Bernhard Hube

Date Published: 29th Jul 2013

Journal: Antimicrob Agents Chemother

Limitation of (1-->3)-beta-D-glucan monitoring in major elective surgery involving cardiopulmonary bypass
FungiNet C - Candida projects

Abstract

Not specified

Authors: G. P. Otto, K. Ludewig, Ilse Jacobsen, Barbara Schaarschmidt, Bernhard Hube, Michael Bauer

Date Published: 12th Jun 2013

Journal: Crit Care

Species and condition specific adaptation of the transcriptional landscapes in Candida albicans and Candida dubliniensis.
FungiNet total, FungiNet C - Candida projects

Abstract (Expand)

BACKGROUND: Although Candida albicans and Candida dubliniensis are most closely related, both species behave significantly different with respect to morphogenesis and virulence. In order to gain further … insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. RESULTS: Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. CONCLUSIONS: Species-specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level.

Authors: C. Grumaz, S. Lorenz, P. Stevens, E. Lindemann, U. Schock, J. Retey, S. Rupp, K. Sohn

Date Published: 4th Apr 2013

Journal: BMC Genomics

Hsp21 potentiates antifungal drug tolerance in Candida albicans
FungiNet C - Candida projects

Abstract (Expand)

Systemic infections of humans with the fungal pathogen Candida albicans are associated with a high mortality rate. Currently, efficient treatment of these infections is hampered by the relatively low … number of available antifungal drugs. We recently identified the small heat shock protein Hsp21 in C. albicans and demonstrated its fundamental role for environmental stress adaptation and fungal virulence. Hsp21 was found in several pathogenic Candida species but not in humans. This prompted us to investigate the effects of a broad range of different antifungal drugs on an Hsp21-null C. albicans mutant strain. Our results indicate that combinatorial therapy targeting Hsp21, together with specific antifungal drug targets, has strong synergistic potential. In addition, we demonstrate that Hsp21 is required for tolerance to ethanol-induced stress and induction of filamentation in response to pharmacological inhibition of Hsp90. These findings might pave the way for the development of new treatment strategies against Candida infections.

Authors: F. L. Mayer, D. Wilson, Bernhard Hube

Date Published: 22nd Mar 2013

Journal: PLoS One

A core filamentation response network in Candida albicans is restricted to eight genes
FungiNet C - Candida projects

Abstract (Expand)

Although morphological plasticity is a central virulence trait of Candida albicans, the number of filament-associated genes and the interplay of mechanisms regulating their expression remain unknown. … By correlation-based network modeling of the transcriptional response to different defined external stimuli for morphogenesis we identified a set of eight genes with highly correlated expression patterns, forming a core filamentation response. This group of genes included ALS3, ECE1, HGT2, HWP1, IHD1 and RBT1 which are known or supposed to encode for cell- wall associated proteins as well as the Rac1 guanine nucleotide exchange factor encoding gene DCK1 and the unknown function open reading frame orf19.2457. The validity of network modeling was confirmed using a dataset of advanced complexity that describes the transcriptional response of C. albicans during epithelial invasion as well as comparing our results with other previously published transcriptome studies. Although the set of core filamentation response genes was quite small, several transcriptional regulators are involved in the control of their expression, depending on the environmental condition.

Authors: R. Martin, Daniela Albrecht-Eckardt, S. Brunke, Bernhard Hube, Kerstin Hünniger, Oliver Kurzai

Date Published: 14th Mar 2013

Journal: PLoS One

Factors supporting cysteine tolerance and sulfite production in Candida albicans
FungiNet C - Candida projects

Abstract (Expand)

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show … that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Delta and ssu1Delta mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Delta mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Authors: F. Hennicke, M. Grumbt, U. Lermann, N. Ueberschaar, K. Palige, B. Bottcher, Ilse Jacobsen, C. Staib, J. Morschhauser, M. Monod, Bernhard Hube, C. Hertweck, P. Staib

Date Published: 15th Feb 2013

Journal: Eukaryot Cell

A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis.
FungiNet C - Candida projects

Abstract (Expand)

Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human … pathogen Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP-Seq and RNA-Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.

Authors: D. Hnisz, A. F. Bardet, C. J. Nobile, A. Petryshyn, W. Glaser, U. Schock, A. Stark, K. Kuchler

Date Published: 14th Dec 2012

Journal: PLoS Genet

An Interspecies Regulatory Network Inferred from Simultaneous RNA-seq of Candida albicans Invading Innate Immune Cells
FungiNet B - Bioinformatics projects, FungiNet C - Candida projects, INF

Abstract (Expand)

The ability to adapt to diverse micro-environmental challenges encountered within a host is of pivotal importance to the opportunistic fungal pathogen Candida albicans. We have quantified C. albicans … and M. musculus gene expression dynamics during phagocytosis by dendritic cells in a genome-wide, time-resolved analysis using simultaneous RNA-seq. A robust network inference map was generated from this dataset using NetGenerator, predicting novel interactions between the host and the pathogen. We experimentally verified predicted interdependent sub-networks comprising Hap3 in C. albicans, and Ptx3 and Mta2 in M. musculus. Remarkably, binding of recombinant Ptx3 to the C. albicans cell wall was found to regulate the expression of fungal Hap3 target genes as predicted by the network inference model. Pre-incubation of C. albicans with recombinant Ptx3 significantly altered the expression of Mta2 target cytokines such as IL-2 and IL-4 in a Hap3-dependent manner, further suggesting a role for Mta2 in host-pathogen interplay as predicted in the network inference model. We propose an integrated model for the functionality of these sub-networks during fungal invasion of immune cells, according to which binding of Ptx3 to the C. albicans cell wall induces remodeling via fungal Hap3 target genes, thereby altering the immune response to the pathogen. We show the applicability of network inference to predict interactions between host-pathogen pairs, demonstrating the usefulness of this systems biology approach to decipher mechanisms of microbial pathogenesis.

Authors: L. Tierney, Jörg Linde, S. Muller, S. Brunke, J. C. Molina, Bernhard Hube, U. Schock, Reinhard Guthke, K. Kuchler

Date Published: 12th Mar 2012

Journal: Front Microbiol

Regulatory interactions for iron homeostasis in Aspergillus fumigatus inferred by a Systems Biology approach
FungiNet A - Aspergillus projects, FungiNet B - Bioinformatics projects, FungiNet C - Candida projects

Abstract (Expand)

BACKGROUND: In System Biology, iterations of wet-lab experiments followed by modelling approaches and model-inspired experiments describe a cyclic workflow. This approach is especially useful for the … inference of gene regulatory networks based on high-throughput gene expression data. Experiments can verify or falsify the predicted interactions allowing further refinement of the network model. Aspergillus fumigatus is a major human fungal pathogen. One important virulence trait is its ability to gain sufficient amounts of iron during infection process. Even though some regulatory interactions are known, we are still far from a complete understanding of the way iron homeostasis is regulated. RESULTS: In this study, we make use of a reverse engineering strategy to infer a regulatory network controlling iron homeostasis in A. fumigatus. The inference approach utilizes the temporal change in expression data after a change from iron depleted to iron replete conditions. The modelling strategy is based on a set of linear differential equations and offers the possibility to integrate known regulatory interactions as prior knowledge. Moreover, it makes use of important selection criteria, such as sparseness and robustness. By compiling a list of known regulatory interactions for iron homeostasis in A. fumigatus and softly integrating them during network inference, we are able to predict new interactions between transcription factors and target genes. The proposed activation of the gene expression of hapX by the transcriptional regulator SrbA constitutes a so far unknown way of regulating iron homeostasis based on the amount of metabolically available iron. This interaction has been verified by Northern blots in a recent experimental study. In order to improve the reliability of the predicted network, the results of this experimental study have been added to the set of prior knowledge. The final network includes three SrbA target genes. Based on motif searching within the regulatory regions of these genes, we identify potential DNA-binding sites for SrbA. Our wet-lab experiments demonstrate high-affinity binding capacity of SrbA to the promoters of hapX, hemA and srbA. CONCLUSIONS: This study presents an application of the typical Systems Biology circle and is based on cooperation between wet-lab experimentalists and in silico modellers. The results underline that using prior knowledge during network inference helps to predict biologically important interactions. Together with the experimental results, we indicate a novel iron homeostasis regulating system sensing the amount of metabolically available iron and identify the binding site of iron-related SrbA target genes. It will be of high interest to study whether these regulatory interactions are also important for close relatives of A. fumigatus and other pathogenic fungi, such as Candida albicans.

Authors: Jörg Linde, P. Hortschansky, E. Fazius, Axel Brakhage, Reinhard Guthke, H. Haas

Date Published: 19th Jan 2012

Journal: BMC Syst Biol

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