Candidalysin crucially contributes to Nlrp3 inflammasome activation by Candida albicans hyphae

Candida albicans is an opportunistic fungal pathogen that can cause life-threatening infections, particularly in immunocompromised patients. C. albicans induced activation of the Nlrp3 inflammasome, leading to secretion of bioactive interleukin 1β (IL-1β) is a crucial myeloid cell immune response needed for antifungal host defense. Being a pleiomorphic fungus, C. albicans can provoke Nlrp3 inflammasome responses only upon morphological transformation to its hyphal appearance. However, the specific hyphal factors that enable C. albicans to activate the Nlrp3 inflammasome in primary macrophages remain to be revealed. Here, we identify candidalysin, a peptide derived from the hypha-specific ECE1 gene, as a fungal trigger for Nlrp3 inflammasome-mediated maturation and secretion of IL-1β from primary macrophages. Direct peptide administration experiments showed that candidalysin was sufficient for inducing secretion of mature IL-1β from macrophages in an Nlrp3 inflammasome-dependent manner. Conversely, infection experiments using candidalysin-deficient C. albicans showed that candidalysin crucially contributed to the capacity of this fungus to induce maturation and secretion of IL-1β from primary macrophages. These complementary observations identify the expression of candidalysin as one of the molecular mechanisms by which hyphal transformation equips C. albicans with its proinflammatory capacity to elicit the release of bioactive IL-1β from macrophages.IMPORTANCE Candidiasis is a potentially lethal condition that is caused by systemic dissemination of Candida albicans, a common fungal commensal residing mostly on mucosal surfaces. The transition of C. albicans from an innocuous commensal to an opportunistic pathogen goes hand in hand with its morphological transformation from a fungus to a hyphal appearance. On the one hand, the latter manifestation enables C. albicans to penetrate tissues, while on the other hand, the expression of many hypha-specific genes also endows it with the capacity to trigger particular cytokine responses. The Nlrp3 inflammasome is a crucial component of the innate immune system that provokes release of the IL-1β cytokine from myeloid cells upon encountering C. albicans hyphae. Our study reveals the peptide candidalysin as one of the hypha-derived drivers of Nlrp3 inflammasome responses in primary macrophages and, thus, contributes to better understanding the fungal mechanisms that determine the pathogenicity of C. albicans

Bioluminescence imaging increases in vivo screening efficiency for antifungal activity against device-associated Candida albicans biofilms

Fungal infections are a major problem for a growing number of mostly immune-compromised patients. Candida albicans is an important human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. The animal models that are currently in use to test antifungal drugs are limited to ex vivo analyses, requiring host sacrifice that excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. As a solution, we introduce non-invasive, dynamic imaging and quantification of C. albicans biofilm formation in vivo and subsequent evaluation of treatment efficacy against these biofilms by using bioluminescent C. albicans in a catheter-associated mouse model. Bioluminescence imaging (BLI) allowed us to evaluate baseline biofilm load before start of therapy, which is necessary for the correct evaluation and interpretation of anti-biofilm efficacy in vivo. Moreover, we demonstrate that our BLI approach monitors the anti-biofilm activity of different antifungal agents efficiently in vitro and in vivo. In this study, BLI revealed superior anti-biofilm activity for echinocandins compared to amphotericin-B and fluconazole. In vitro, anidulafungin showed the highest anti-biofilm activity, followed by micafungin and caspofungin. In vivo, caspofungin significantly decreased the biofilm fungal load, as documented by the lower BLI signal and confirmed by CFU counts. In conclusion, our BLI approach increases the power and efficiency of screening and validation of antimycotics under both in vitro and in vivo conditions, thereby refining preclinical therapy studies.status: publishe

In Vivo Efficacy of Anidulafungin against Mature Candida albicans Biofilms in a Novel Rat Model of Catheter-Associated Candidiasis▿

The present study demonstrates the efficacy of anidulafungin on mature Candida albicans biofilms in vivo. One hundred fifty-seven catheter fragments challenged with C. albicans were implanted subcutaneously in rats. After formation of biofilms, rats were treated with daily intraperitoneal injections of anidulafungin for 7 days. Catheters retrieved from treated animals showed reduced cell numbers compared to those retrieved from untreated and fluconazole-treated animals. Systemic administration of anidulafungin is promising for the treatment of mature C. albicans biofilms

Real-time PCR expression profiling of genes encoding potential virulence factors in <it>Candida albicans </it>biofilms: identification of model-dependent and -independent gene expression

<p>Abstract</p> <p>Background</p> <p><it>Candida albicans </it>infections are often associated with biofilm formation. Previous work demonstrated that the expression of <it>HWP1 </it>(hyphal wall protein) and of genes belonging to the <it>ALS </it>(agglutinin-like sequence), <it>SAP </it>(secreted aspartyl protease), <it>PLB </it>(phospholipase B) and <it>LIP </it>(lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, <it>C. albicans </it>biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.</p> <p>Results</p> <p><it>HWP1 </it>and genes belonging to the <it>ALS</it>, <it>SAP</it>, <it>PLB </it>and <it>LIP </it>gene families were constitutively expressed in <it>C. albicans </it>biofilms. <it>ALS1-5 </it>were upregulated in all model systems, while <it>ALS9 </it>was mostly downregulated. <it>ALS6 </it>and <it>HWP1 </it>were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of <it>SAP1 </it>were more pronounced in both in vitro models, while those of <it>SAP2</it>, <it>SAP4 </it>and <it>SAP6 </it>were higher in the in vivo model. Furthermore, <it>SAP5 </it>was highly upregulated in the in vivo and RHE models. For <it>SAP9 </it>and <it>SAP10 </it>similar gene expression levels were observed in all model systems. <it>PLB </it>genes were not considerably upregulated in biofilms, while <it>LIP1-3, LIP5-7 </it>and <it>LIP9</it>-<it>10 </it>were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.</p> <p>Conclusions</p> <p>Our findings show that <it>HWP1 </it>and most of the genes belonging to the <it>ALS, SAP </it>and <it>LIP </it>gene families are upregulated in <it>C. albicans </it>biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in <it>C. albicans </it>biofilms is highlighted.</p

Antifungal activity of oleylphosphocholine on in vitro and in vivo Candida albicans biofilms

In this study, we investigated the potential antifungal activity of the alkylphospholipid - oleylphosphocholine (OlPC), a structural analogue of miltefosine, on in vitro and in vivo Candida albicans biofilm formation. The effect of OlPC on in vitro and in vivo C. albicans biofilms was studied inside triple-lumen polyurethane catheters. In vivo biofilms were developed subcutaneously after catheter implant on the lower back of Sprague Dawley rats. Animals were treated orally with OlPC (20 mg/kg of body weight/day) for 7 days. The effect of OlPC on biofilms developed on mucosal surface was studied in an ex vivo model of oral candidiasis. The role of OlPC on C. albicans morphogenesis was investigated in hyphae-inducing media namely, Lee, Spider and RPMI 1640. OlPC displayed activity against both planktonic cells and in vitroC. albicans biofilms. To completely abolish preformed, 24 h old biofilms, higher concentrations (8, 10 and 13 mg/L) were needed. Moreover, OlPC was able to reduce C. albicans biofilms formed by caspofungin-resistant clinical isolates and acted synergistically when combined with caspofungin. Daily administration of OlPC orally, significantly reduced in vivo C. albicans biofilms developed subcutaneously. In addition, OlPC decreased biofilm formation on mucosal surfaces. Interestingly, application of sub-inhibitory concentrations of OlPC already inhibited the yeast-to-hyphae transition, a crucial virulence factor of C. albicans We document, for the first time, the effects of OlPC on C. albicans cells and suggest the potential use of OlPC in the treatment of C. albicans biofilm-associated infections.status: publishe

Anidulafungin increases the antibacterial activity of tigecycline in polymicrobial Candida albicans/Staphylococcus aureus biofilms on intraperitoneally implanted foreign bodies

Objectives: We aimed to establish a novel murine intra-abdominal foreign body infection model to study the activity of anidulafungin and tigecycline against dual species Candida albicans/Staphylococcus aureus biofilms. Methods: In vitro and in vivo single and dual species biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well plates or implanted intraperitoneally in BALB/c mice. The effect of tigecycline and anidulafungin alone and in combination was tested using clinically relevant concentrations. Scanning electron microscopy was used to visualize the mature biofilm structure developed intraperitoneally. Flow cytometry was used to determine the immunological response upon infection. Immunoblot analysis allowed us to determine the effect of anidulafungin on poly-β-(1,6)-N-acetylglucosamine in in vitro-grown S. aureus biofilms. Results: We determined the MIC, MBC and in vitro susceptibility profile for anidulafungin and tigecycline against C. albicans and S. aureus in mixed and single species biofilms. We demonstrated that anidulafungin acts synergistically when combined with tigecycline against in vivo intra-abdominal biofilms. Moreover, we reveal that anidulafungin reduces the abundance of S. aureus poly-β-(1,6)-N-acetylglucosamine. The influx of neutrophils is much increased when infected with mixed biofilms compared with single species biofilms. Conclusions: Currently, treatment of intra-abdominal infections, in particular polymicrobial catheter-associated peritonitis, is ineffective. To the best of our knowledge, this is the first study that provides insight into new possible options for treatment of C. albicans/S. aureus biofilms present in the abdominal cavity