The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.

SummaryThe cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/ pop2 mutants display reduced wall b-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans . We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe , such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans , filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans : they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans . This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species.

Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription.C ELL-wall adhesins mediate attachment between cells and to abiotic substrates. Adhesion is key for the ability of unicellular yeasts to change morphology, mate, invade substrates and host cells, and associate into protective multicellular communities, such as biofilms and flocs (reviewed in Bruckner and Mosch 2012). These pathways are important for industrial applications and in the context of human disease caused by fungal pathogens (Hoyer et al. 2008;Finkel and Mitchell 2011;Liu and Filler 2011; Bruckner and Mosch 2012). In the model yeast Saccharomyces cerevisiae, the cell-wall adhesin Flo11 mediates many such adhesion-related phenotypes, such as attachment to polystyrene and formation of multicellular structures called "mats" (Lo and Dranginis 1998;Guo et al. 2000;Reynolds and Fink 2001). Mats form when yeast cells spread over a semisolid agar substrate and have a defined "floral" architecture, suggestive of a developmental pathway (Reynolds and Fink 2001). The regulation of the expression of the FLO11 gene has long served as a model for understanding how extracellular signals, developmental pathways, and epigenetic mechanisms impinge on gene expression (reviewed in Bruckner and Mosch 2012). The expression of FLO11 depends on the environmental context (e.g., nutrient limitation, quorum sensing) and is regulated by a range of transcriptional activators and repressors under the control of signaling pathways. For example, the cAMP/PKA pathway, the mitogen-activated protein kinase pathway, and the pH-responsive Rim101 pathway all regulate the expression of FLO11 through transcriptional activators such as Flo8, Ste12, Tec1, and Rme1 and repressors such as Sfl1, Nrg1, Nrg2, and Sok2 (Lo and Dranginis 1998;Kuchin et al. 2002; Braus et al. 2003;Vyas et al. 2003;Chen and Fink 2006; Bruckner and Mosch 2012). Epigenetic mechanisms also control FLO11 expression (Halme et al. 2004).The mRNA decay pathway is important for the control of mRNA stability and translation (Goldstrohm and Wickens 2008). The components of this pathway include the Ccr4-NOT mRNA deadenylase complex, which shortens the mRNA poly(A) tail as the first step leading to mRNA decay; the decapping enzyme Dcp1/Dcp2 catalyzing 59 cap hydrolysis following deadenylation; the exonuclease Xrn1 that degrades the mRNA after decapping; and decapping activators such as the RNA helicase Dhh1 (Parker and Song 2004;Goldstrohm and Wickens 2008). While deadenylation and decay act on all transcripts during their life cycle, it is thought that additional, gene-specific effects also occur (Beilharz and Preiss 2007;Lackner et al. 2007). This is manifested in transcripts displaying different steady-state distribution of poly(A) ...

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

15Peritonitis is a leading cause of severe sepsis in surgical intensive care units, as 16 over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of 17 65 non-mucosal fungal diseases among hospitalized patients in the developed world are 66Candida peritonitis, also referred to as intra-abdominal candidiasis (IAC). IAC is 67 challenging to diagnose and hence results in high mortality rates ranging from 25% -68

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