Fungal Signal Research Articles

Clinical and Radiologic Features of Fungal Diseases of the Paranasal Sinuses

To document the clinical and imaging characteristics of fungal rhinosinusitis (FRS). Imaging studies of 28 patients with FRS were retrospectively analyzed, considering the type of fungal disease, location, signal characteristics, bone changes, expansion, and extrasinus extension. Acute invasive FRS showed unilateral pacifications of the sinonasal cavity, perisinus fat infiltration and/or bone destruction. Chronic invasive FRS demonstrated masslike hyperattenuating soft tissue, with bony destruction. The soft tissue changes were hypointense on T1 and markedly hypointense on T2-weighted images. In allergic FRS, hyperattenuating soft tissue causing paranasal expansion due to allergic mucin was observed on CT. Fungus ball presented as a hyperattenuating lesion with calcifications within a single sinus. The radiological features of each type of FRS are distinctive and should afford a specific diagnosis in the proper clinical setting.

Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses

C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.

Know your enemy: how to build and vanquish a global fungal scourge.

The 8th International Conference on Cryptococcus and Cryptococcosis, chaired by Maurizio Del Poeta (Medical University of South Carolina), and organized together with June Kwon-Chung (National Institute of Allergy and Infectious Diseases), Stuart Levitz (University of Massachusetts Medical School), and John Perfect (Duke University), occurred in May 2011. This meeting brought together the world's leading researchers on Cryptococcus and cryptococcosis, including basic scientists, epidemiologists, and clinicians, to discuss new developments in Cryptococcus biology. With more than 60 oral presentations and 180 posters, this meeting enhanced our understanding of pathogenicity of Cryptococcus and served as a robust forum that facilitated cross-disciplinary discussions, research, and clinical collaborations. Due to space constraints, this brief overview highlights only a few of the topics discussed in this meeting, focusing on the evolution of virulence, host and pathogen interactions, fungal and host signaling, new advances of genomics studies on Cryptococcus, and the current status of the outbreak caused by C. gattii. The 8th International Conference on Cryptococcus and Cryptococcosis brought together scientists from across the globe in the beautiful historical downtown setting of Charleston to share their latest findings and highlight advances in Cryptococcus research. With more than 250 participants, this meeting was the largest gathering of the Cryptococcus international community in the 24-year history. Here, we review the advances presented and the current state of knowledge in the field.

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation.

Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.

Root starch accumulation in response to arbuscular mycorrhizal colonization differs among Lotus japonicus starch mutants

Arbuscular mycorrhizal (AM) fungi are obligate symbionts dependent for completion of their life cycle on plant carbohydrates, which they trade for mineral nutrients. Plant colonization by AM fungi is therefore expected to induce profound changes in plant carbon metabolism. We have previously observed that on one hand starch accumulation increases in responses to pre-symbiotic fungal signals and on the other hand, it decreases in mycorrhizal Lotus japonicus roots (Gutjahr et al. in New Phytol 183:53-61, 2009). To examine the importance of starch metabolism for AM development, we took advantage of a novel series of Lotus japonicus mutants impaired either in starch degradation or in synthesis. Normal AM colonization in all mutants indicated that defects in starch metabolism do not affect AM development and that carbohydrates can be supplied to the AM fungus without a requirement for starch synthesis. Furthermore, our experiments allowed us to characterize root starch dynamics in detail and point to continued turnover of starch in the degradation mutants in the presence of mycorrhiza.

Transcriptome analysis shows differential gene expression in the saprotrophic to parasitic transition of Pochonia chlamydosporia

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.

Unlocking the Door to Invasion

A single plant receptor recognizes related bacterial and fungal signals to initiate symbiosis.

Regulators of G‐protein signalling in Fusarium verticillioides mediate differential host–pathogen responses on nonviable versus viable maize kernels

GBB1, a heterotrimeric G-protein β-subunit gene, was shown to be a key regulator of fumonisin B(1) (FB(1) ) biosynthesis in the maize pathogen Fusarium verticillioides. In this study, we performed functional analyses of genes that encode putative RGS (regulators of G-protein signalling) proteins and PhLPs (phosducin-like proteins) in F. verticillioides. These proteins are known to regulate heterotrimeric G-protein activity by altering the intrinsic guanosine triphosphatase (GTPase) activity, which, in turn, influences the signalling mechanisms that control fungal growth, virulence and secondary metabolism. Our aim was to isolate and characterize gene(s) that are under the transcriptional control of GBB1, and to test the hypothesis that these genes are directly associated with FB(1) regulation and fungal development in F. verticillioides on maize kernels. We first identified eight genes (two PhLPs and six RGSs) in the F. verticillioides genome, and a subsequent transcriptional expression study revealed that three RGS genes were up-regulated in the gbb1 deletion (Δgbb1) mutant and one RGS gene was up-regulated in the wild-type. To characterize their function, we generated knockout mutants using a homologous recombination strategy. When grown on autoclaved nonviable kernels, two mutants (ΔflbA2 and ΔrgsB) produced significantly higher levels of FB(1) compared with the wild-type progenitor, suggesting that the two mutated genes are negative regulators of FB(1) biosynthesis. ΔflbA2 also showed a severe curly conidia germination pattern, which was contradictory to that observed in the Δgbb1 strain. Strikingly, when these mutants were grown on live maize kernels, we observed contrasting FB(1) and conidiation phenotypes in fungal mutants, which strongly suggests that these G-protein regulators have an impact on how F. verticillioides responds to host/environmental factors. Our data also provide evidence that fungal G-protein signalling is important for modulating the ethylene biosynthetic pathway in maize kernels.

Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca2+ spiking in the legume and nonlegume root epidermis

• The aim of this study was to investigate Ca(2+) responses to endosymbiotic arbuscular mycorrhizal (AM) fungi in the host root epidermis following pre-infection hyphopodium formation in both legumes and nonlegumes, and to determine to what extent these responses could be mimicked by germinated fungal spore exudate. • Root organ cultures of both Medicago truncatula and Daucus carota, expressing the nuclear-localized cameleon reporter NupYC2.1, were used to monitor AM-elicited Ca(2+) responses in host root tissues. • Ca(2+) spiking was observed in cells contacted by AM hyphopodia for both hosts, with highest frequencies correlating with the epidermal nucleus positioned facing the fungal contact site. Treatment with AM spore exudate also elicited Ca(2+) spiking within the AM-responsive zone of the root and, in both cases, spiking was dependent on the M. truncatula common SYM genes DMI1/2, but not on the rhizobial Nod factor perception gene NFP. • These findings support the conclusion that AM fungal root penetration is preceded by a SYM pathway-dependent oscillatory Ca(2+) response, whose evolutionary origin predates the divergence between asterid and rosid clades. Our results further show that fungal symbiotic signals are already generated during spore germination, and that cameleon-expressing root organ cultures represent a novel AM-specific bio-assay for such signals.

TheRIM101Signal Transduction Pathway RegulatesCandida albicansVirulence during Experimental Keratomycosis

To examine the role of the fungal RIM101 signal transduction pathway in the pathogenesis of Candida albicans keratitis. C. albicans wild-type strain SC5314, prototrophic mutant control DAY185, and homozygous fungal mutants for the rim8, rim13, rim20, rim101, and phr1 genes were evaluated in vitro using proliferation and filamentation assays. Scarified corneas of BALB/c and C57BL/6J mice were topically inoculated and observed daily for keratitis severity. Corneal adaptation and pathogenicity were assessed ex vivo by maintaining infected porcine corneas for 3 days in an explantation culture system for histologic evaluation of hyphal penetration. All C. albicans strains had similar growth kinetics, and SC5314 and DAY185 demonstrated pH-induced filamentation. Fungal mutants had reduced hyphal formation at alkaline and neutral pH, but normal acidic assays ascertained that mutant strains did not have a generalized filamentation defect. SC5314 and DAY185 caused moderate to severe keratitis in mice, whereas fungal strains lacking constituents of the RIM101 pathway had significantly (P<0.05) attenuated severity in vivo. Three days after inoculation of porcine corneas, SC5314 and DAY185 produced hyphae that penetrated 28% and 25%, respectively, of the corneal thickness, and all five mutant strains showed significantly (P<0.05) less stromal penetration. The RIM101 signal transduction pathway plays an important role in the development of C. albicans keratitis. The fungal pathway intermediates Rim8p, Rim13p, Rim20p, and Rim101p and the downstream cell-wall protein Phr1p are pivotal in the process of corneal invasion by C. albicans.

Medullosalean fusain trunk from the roof rocks of a coal seam: Insight from FTIR and NMR (Pennsylvanian Sydney Coalfield, Canada)

Reported for the first time from the Sydney Coalfield, Canada, is a fragmentary fusain (Ro = 2.51%) specimen, 41 cm long, of a medullosalean trunk or massive petiole that originated from the roof rocks of the banded bituminous Hub Seam (Ro ∼ 0.65%). Megascopic characteristics of the flat-preserved specimen include an irregular-cracked fusain surface with secretinite–rodlet structures and sclerenchymatous strands some of which still embedded in the shaly matrix, and locally preserved vitrain (Ro = 0.69%). Co-occurrence with a compression flora composed virtually of only the seed-fern taxon Macroneuropteris scheuchzeri (Hoffmann) is noted. The goal of this study is to provide a framework for the phytophysicochemical taphonomic history which includes the perspective on vitrinite/fusinite relationship, formation of fusinite, and on the significance the only fusinized identifiable plant-fossil specimen in the Sydney Coalfield. We use state-of-the-art solid-state FTIR, 13C, 1H NMR CP/MAS techniques, and standard reflected-light microscopy and SEM methods as investigative tools. Results indicate that the fusinite is characterized by long and narrow xylem fibers, without fungal signals, and cell structures infilled with pyrite and carbonate. FTIR spectra of the fusinite and secretinite are similar particularly in respect to high absorbance of aromatic and low absorbance of aliphatic compounds, and absorbance of Si–O functionalities relating to kaolinite. 13C NMR experiments with direct carbon excitation quantify the aromatic to aliphatic ratio as being 20 ± 3:1. As part of the taphonomic history, the evidence favors a hot, > 400 °C, directional surface-fueled flame palaeofire of local extent that charred trunks of growing arborescent seed ferns on only one side, and that these trunks were transported to comprise part of the roof rocks of the Hub Seam.

Recruitment of the ESCRT Machinery to a Putative Seven-Transmembrane-Domain Receptor Is Mediated by an Arrestin-Related Protein

Mammalian arrestins have a major role in the intracellular trafficking of seven-transmembrane (7TM) receptors. The fungal ambient pH signaling pathway involves an arrestin-related protein, PalF/Rim8, and the ESCRT (endosomal sorting complex required for transport) machinery. We found that in Saccharomyces cerevisiae, Rim8 binds to both the putative 7TM pH sensor Rim21 and the ESCRT-I subunit Vps23. We show that an SXP motif in Rim8 mediates binding to the Vps23 ubiquitin E2 variant (UEV) domain and that a monoubiquitinated residue near the SXP motif contributes to this interaction. We present evidence that Rim8 ubiquitination is dependent on the Rsp5 E3 ubiquitin ligase and triggered upon binding of Vps23 UEV to both the SXP motif and ubiquitin, thus suggesting a two-step binding mechanism. We further show that Rim8 coimmunoprecipitates with ESCRT-I subunits Vps23 and Vps28, supporting the idea that binding of Rim8 to Vps23 mediates the association of Rim8 with the ESCRT-I complex. Fluorescence microscopic analyses indicate that overexpressed Rim8 and Vps23 colocalize at cortical punctate structures, providing additional evidence of the interaction between these two proteins. Strikingly, our findings indicate that evolutionary conserved mechanisms control the recruitment of the ESCRT machinery to Pal/Rim proteins in fungi and retroviral Gag proteins in animal cells.

Farnesol Induces Hydrogen Peroxide Resistance in Candida albicans Yeast by Inhibiting the Ras-Cyclic AMP Signaling Pathway

Farnesol, a Candida albicans cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, alpha-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate that farnesol induces oxidative-stress resistance by a similar mechanism. Strains lacking either Ras1 or Cyr1 no longer exhibited increased protection against hydrogen peroxide upon preincubation with farnesol. While we also observed the previously reported increase in the phosphorylation level of Hog1, a known regulator of oxidative-stress resistance, in the presence of farnesol, the hog1/hog1 mutant did not differ from wild-type strains in terms of farnesol-induced oxidative-stress resistance. Analysis of Hog1 levels and its phosphorylation states in different mutant backgrounds indicated that mutation of the components of the Ras1-adenylate cyclase pathway was sufficient to cause an increase of Hog1 phosphorylation even in the absence of farnesol or other exogenous sources of oxidative stress. This finding indicates the presence of unknown links between these signaling pathways. Our results suggest that farnesol effects on the Ras-adenylate cyclase cascade are responsible for many of the observed activities of this fungal signaling molecule.

Candida albicans β-Glucan Exposure Is Controlled by the Fungal CEK1 -Mediated Mitogen-Activated Protein Kinase Pathway That Modulates Immune Responses Triggered through Dectin-1

Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as beta-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall beta-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IkappaB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in beta-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

Microarray analysis and functional tests suggest the involvement of expansins in the early stages of symbiosis of the arbuscular mycorrhizal fungus Glomus intraradices on tomato (Solanum lycopersicum).

Arbuscular mycorrhizal (AM) symbiosis occurs between fungi of the phylum Glomeromycota and most terrestrial plants. However, little is known about the molecular symbiotic signalling between AM fungi (AMFs) and non-leguminous plant species. We sought to further elucidate the molecular events occurring in tomato, a non-leguminous host plant, during the early, pre-symbiotic stage of AM symbiosis, i.e. immediately before and after contact between the AMF (Glomus intraradices) and the host. We adopted a semi-synchronized AMF root infection protocol, followed by genomic-scale, microarray-based, gene expression profiling at several defined time points during pre-symbiotic AM stages. The microarray results suggested differences in the number of differentially expressed genes and in the differential regulation of several functional groups of genes at the different time points examined. The microarray results were validated and one of the genes induced during contact between AMF and tomato, the expansin-like EXLB1, was functionally analysed. Expansins, encoded by a large multigene family, facilitate plant cell expansion. However, no biological or biochemical function has yet been established for plant-originated expansin-like proteins. EXLB1 transcripts were localized early during the association to cells that may perceive the fungal signal, and later during the association in close proximity to sites of AMF hypha-root colonization. Moreover, in transgenic roots, we demonstrated that a reduction in the steady-state level of EXLB1 transcript was correlated with a reduced rate of infection, reduced arbuscule expansion and reduced AMF spore formation.

Membrane steroid‐binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula

Arbuscular mycorrhiza (AM) is a mutualistic biotrophic association that requires a complex exchange of signals between plant and fungus to allow accommodation of the mycosymbiont in the root cortex. Signal exchange happens even before physical contact, activating the plant symbiotic program. We investigated very early transcriptional responses in Medicago truncatula to inoculation with Glomus intraradices and identified four genes induced by diffusible AM fungal signals before contact. Three of them were previously shown to be mycorrhiza induced at later stages of the symbiosis, while MtMSBP1, encoding a membrane-bound steroid-binding protein, is a novel mycorrhizal marker. Expression analyses in plants defective in the symbiotic receptor kinase DMI2 allowed discrimination of two different signaling cascades involved in the perception of the diffusible signals. Thus, while some of the genes are activated in a DMI2-dependent manner, the induction of one of them encoding a proteinase inhibitor is DMI2-independent. Downregulation of MtMSBP1 by RNAi led to an aberrant mycorrhizal phenotype with thick and septated appressoria, decrease number of arbuscules and distorted arbuscule morphology. This provides genetic evidence that MtMSBP1 is critical for mycorrhiza development. We hypothesize that MtMSBP1 plays a role in sterol homeostasis in the root.

Fungal physiology and the origins of molecular biology

Molecular biology has several distinct origins, but especially important are those contributed by fungal and yeast physiology, biochemistry and genetics. From the first gene action studies that became the basis of our understanding of the relationship between genes and proteins, through chromosome structure, mitochondrial genetics and membrane biogenesis, gene silencing and circadian clocks, studies with these organisms have yielded basic insight into these processes applicable to all eukaryotes. Examples are cited of pioneering studies with fungi that have stimulated new research in clinical medicine and agriculture; these studies include sexual interactions, cell stress responses, the cytoskeleton and pathogenesis. Studies with the yeasts and fungi have been effective in applying the techniques and insights gained from other types of experimental systems to research in fungal cell signalling, cell development and hyphal morphogenesis.

A Fungal pH-Responsive Signaling Pathway RegulatingAspergillusAdaptation and Invasion into the Cornea

To investigate the role of PalB and PacC, two components of a pH-responsive signal-transduction pathway of Aspergillus nidulans, during the pathogenesis of fungal infection of the cornea. Fungal strains included an A. nidulans wild-type isolate (A83), loss-of-function A. nidulans mutants of the palB (B7) or pacC (C6309) genes, and reconstituted genotypic strains (B7R and C6309R). Doubling times and radial growth rates were examined under neutral and acidic conditions. Corneal virulence was assessed ex vivo by topical inoculation of scarified porcine or human corneas with A. nidulans strains maintained in buffered medium until histologic examination after days 1, 3, and 5. In vitro growth kinetics were similar for A. nidulans strains in liquid medium at pH 6.0 (P = 0.24) and 7.3 (P = 0.75). The pacC mutant C6309 grew more slowly (P < 0.001) on solid medium, whereas palB and pacC rescuants had growth kinetics comparable to those of the wild-type. Wild-type A. nidulans germinated on porcine corneas and produced hyphae that progressively invaded the stroma, reaching an average maximum penetration of 56% +/- 9% at 5 days after exposure. In contrast, hyphal invasion was significantly less by mutant strains B7 (P = 0.005) and C6309 (P = 0.003). Fungal penetration by C6309 was also significantly less than the wild-type (P = 0.0005) on explanted human corneas. Both fungal rescuants showed stromal invasion similar to the wild-type. Corneal invasion by filamentous hyphae is attenuated by palB and pacC mutant strains of A. nidulans. The PacC pathway is involved in regulating fungal filamentation during ex vivo Aspergillus infection of the cornea.

Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence.

Fungal infections represent a serious threat, particularly in immunocompromised patients. Interleukin-1beta (IL-1beta) is a key pro-inflammatory factor in innate antifungal immunity. The mechanism by which the mammalian immune system regulates IL-1beta production after fungal recognition is unclear. Two signals are generally required for IL-1beta production: an NF-kappaB-dependent signal that induces the synthesis of pro-IL-1beta (p35), and a second signal that triggers proteolytic pro-IL-1beta processing to produce bioactive IL-1beta (p17) via Caspase-1-containing multiprotein complexes called inflammasomes. Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1beta synthesis and inflammasome activation after cell stimulation with Candida albicans. Whereas Syk signalling for pro-IL-1beta synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1beta processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1beta production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo.

NLRP12 REGULATES TLR2/DECTIN-1 FUNGAL RECEPTOR SIGNALING THROUGH ASSOCIATION WITH CARD9 (135.74)

Abstract The NLR (nucleotide-binding domain, leucine-rich repeat) proteins are recently recognized as important intracellular regulators of host defense and immunity. One NLR gene, NLRP12/Monarch-1, has emerged as an important inhibitor of inflammatory gene expression in myeloid cells via regulation of TLRs and TNFR signal transduction. In contrast to its known role in regulating bacterial induced TLR activation of downstream genes, little is known about the role of NLRP12 in regulating the immune response to fungal pathogens. Here we show that NLRP12 modulates innate immune cellular responses to A. fumigatus by regulating TLR2/Dectin-1 fungal receptor signaling through its association with downstream signaling proteins CARD9 and Bcl10, but not Malt1. NLRP12, consisting of a pyrin, a nucleotide binding and a leucine rich repeat domain associates with the caspase recruitment domain containing proteins CARD9 and Bcl10 via the adaptor protein ASC. The most severe form of the NLRP12 disease associated mutant protein lacks the ability to associate with ASC, CARD9 and Bcl10 resulting in the lack of NLRP12's normal negative regulatory function. These data demonstrate the important role played by NLRP12 in modulating the innate immune response to fungal pathogens via its regulation of the TLR2/Dectin-1 induced CARD9 signaling pathway. This work was supported by NIH grants HL-30923 and HL-68072 (JRW), NIH COBRE grant RR020185 (RAC) a SERCEB award and funding from CCBVP to KLW.