Candida albicans Hyphal Extracellular Vesicles Are Different from Yeast Ones, Carrying an Active Proteasome Complex and Showing a Different Role in Host Immune Response.

Candida albicans is the principal causative agent of lethal fungal infections, predominantly in immunocompromised hosts. Extracellular vesicles (EVs) have been described as crucial in the interaction of microorganisms with their host. Since the yeast-to-hypha transition is an important virulence trait with great impact in invasive candidiasis (IC), we have addressed the characterization of EVs secreted by hyphal cells (HEVs) from C. albicans, comparing them to yeast EVs (YEVs). YEVs comprised a larger population of bigger EVs with mainly cell wall proteins, while HEVs were smaller, in general, and had a much higher protein diversity. YEVs were able to rescue the sensitivity of a cell wall mutant against calcofluor white, presumably due to the larger amount of cell wall proteins they contained. On the other hand, HEVs also contained many cytoplasmic proteins related to protein metabolism and intracellular protein transport and the endosomal sorting complexes required for transport (ESCRT) pathway related to exosome biogenesis, pointing to an intracellular origin of HEVs. Interestingly, an active 20S proteasome complex was secreted exclusively in HEVs. Moreover, HEVs contained a greater number of virulence-related proteins. As for their immunogenic role, both types of EV presented immune reactivity with human sera from patients suffering invasive candidiasis; however, under our conditions, only HEVs showed a cytotoxic effect on human macrophages and could elicit the release of tumor necrosis factor alpha (TNF-α) by these macrophages. IMPORTANCE This first analysis of HEVs of C. albicans has shown clear differences between them and the YEVs of C. albicans, showing their relevance and possible use in the discovery of new diagnostic markers and treatment targets against C. albicans infections. The data obtained point to different mechanisms of biogenesis of YEVs and HEVs, as well as different involvements in cell biology and host interaction. YEVs played a more relevant role in cell wall maintenance, while HEVs were more closely related to virulence, as they had greater effects on human immune cells. Importantly, an active 20S proteosome complex was described as a fungal-EV cargo. A deeper study of its role and those of many other proteins exclusively detected in HEVs and involved in different relevant biological processes of this fungus could open up interesting new areas of research in the battle against C. albicans.

Association of Immune Cell Subtypes and Phenotype With Subsequent Invasive Candidiasis in Patients With Abdominal Sepsis.

BACKGROUND: In nonneutropenic intensive care unit (ICU) patients, current risk stratification scores lack specificity to reliably predict the risk of a prospective invasive candidiasis (IC). We aimed to explore possible associations of distinct immunological markers with different degrees of Candida affection in patients with abdominal sepsis. METHODS: The presented explorative, noninterventional diagnosis study recruited patients admitted to the surgical ICU at Heidelberg University Hospital with abdominal sepsis. Over 5 days, we determined white blood cell count, 1,3-ß-D-glucan, and HLA-DR expression; the amount of Th1, Th17, regulatory T, T helper, and cytotoxic T cells; Dectin-1 and TLR2-expression; the amount of T, B, and NK cells; the ex vivo secretion of IL-8 upon stimulation with LPS, flagellin, and zymosan; and the distribution of distinct T-cell cytokines in a daily manner. On day 21, patients' Candida infection status was stratified in no colonization or IC, colonization or IC. RESULTS: A total of 26 patients were included. On day 21, five patients showed no colonization or IC, in 13 patients a colonization was detected, and eight patients were diagnosed with IC. On study inclusion, the stratification groups showed comparable values in standard laboratory parameters and morbidity scores. Decreased B and NK cell counts, as well as reduced IL-8 secretion after ex vivo stimulation with LPS or flagellin seemed to be associated with a higher risk of subsequent Candida colonization. Even lower values could distinguish the therapy-relevant difference between prospective IC from colonization alone. CONCLUSIONS: We were able to show distinct immune system impairments in early abdominal sepsis by specific immune-based measurements. A possible association of these impairments with a subsequent Candida affection is shown.

Significantly Improved Pharmacokinetics Enhances In Vivo Efficacy of APX001 against Echinocandin- and Multidrug-Resistant Candida Isolates in a Mouse Model of Invasive Candidiasis.

APX001 is a first-in-class, intravenous and orally available, broad-spectrum antifungal agent in clinical development for the treatment of life-threatening invasive fungal infections. The half-life of APX001A, the active moiety of APX001, is significantly shorter in mice than in humans (1.4 to 2.75 h in mice versus 2 to 2.5 days in humans), making the exploration of efficacy in mouse models difficult. After pretreatment with 1-aminobenzotriazole (ABT), a nonspecific cytochrome P450 inhibitor, greatly increased plasma APX001A exposure was observed in mice of different strains and of both genders. As a consequence, 26 mg/kg APX001 plus ABT sterilized kidneys in mice infected with Candida albicans, while APX001 alone at the same dose resulted in a modest burden reduction of only 0.2 log10 CFU/g, relative to the vehicle control. In the presence of ABT, 2 days of once-daily dosing with APX001 at 26 mg/kg also demonstrated significant in vivo efficacy in the treatment of Candida glabrata infections in mice. Potent kidney burden reduction was achieved in mice infected with susceptible, echinocandin-resistant, or multidrug-resistant strains. In contrast, the standard of care (micafungin) was ineffective in treating infections caused by the resistant C. glabrata isolates.

Biphasic zinc compartmentalisation in a human fungal pathogen.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Investigation of Candida parapsilosis virulence regulatory factors during host-pathogen interaction.

Invasive candidiasis is among the most life-threatening infections in patients in intensive care units. Although Candida albicans is the leading cause of candidaemia, the incidence of Candida parapsilosis infections is also rising, particularly among the neonates. Due to differences in their biology, these species employ different antifungal resistance and virulence mechanisms and also induce dissimilar immune responses. Previously, it has been suggested that core virulence effecting transcription regulators could be attractive ligands for future antifungal drugs. Although the virulence regulatory mechanisms of C. albicans are well studied, less is known about similar mechanisms in C. parapsilosis. In order to search for potential targets for future antifungal drugs against this species, we analyzed the fungal transcriptome during host-pathogen interaction using an in vitro infection model. Selected genes with high expression levels were further examined through their respective null mutant strains, under conditions that mimic the host environment or influence pathogenicity. As a result, we identified several mutants with relevant pathogenicity affecting phenotypes. During the study we highlight three potentially tractable signaling regulators that influence C. parapsilosis pathogenicity in distinct mechanisms. During infection, CPAR2_100540 is responsible for nutrient acquisition, CPAR2_200390 for cell wall assembly and morphology switching and CPAR2_303700 for fungal viability.

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis.

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.

Initiation of phospholipomannan ß-1,2 mannosylation involves Bmts with redundant activity, influences its cell wall location and regulates ß-glucans homeostasis but is dispensable for Candida albicans systemic infection.

Pathogenic and non-pathogenic fungi synthesize glycosphingolipids, which have a crucial role in growth and viability. Glycosphingolipids also contribute to fungal-associated pathogenesis. The opportunistic yeast pathogen Candida albicans synthesizes phospholipomannan (PLM), which is a glycosphingolipid of the mannosylinositol phosphorylceramide family. Through its lipid and glycan moieties, PLM contributes to the initial recognition of the yeast, causing immune system disorder and persistent fungal disease through activation of host signaling pathways. The lipid moiety of PLM activates the deregulation signaling pathway involved in yeast phagocytosis whereas its glycan moiety, composed of ß-1,2 mannosides (ß-Mans), participates to inflammatory processes through a mechanism involving Galectin-3. Biosynthesis of PLM ß-Mans involves two ß-1,2 mannosyltransferases (Bmts) that initiate (Bmt5) and elongate (Bmt6) the glycan chains. After generation of double bmtsΔ mutants, we show that Bmt5 has redundant activity with Bmt2, which can replace Bmt5 in bmt5Δ mutant. We also report that PLM is located in the inner layer of the yeast cell wall. PLM seems to be not essential for systemic infection of the yeast. However, defect of PLM ß-mannosylation increases resistance of C. albicans to inhibitors of ß-glucans and chitin synthesis, highlighting a role of PLM in cell wall homeostasis.

MicroRNA Expression Profiling of Human Respiratory Epithelium Affected by Invasive Candida Infection.

Invasive candidiasis is potentially life-threatening systemic fungal infection caused by Candida albicans (C. albicans). Candida enters the blood stream and disseminate throughout the body and it is often observed in hospitalized patients, immunocompromised individuals or those with chronic diseases. This infection is opportunistic and risk starts with the colonization of C. albicans on mucocutaneous surfaces and respiratory epithelium. MicroRNAs (miRNAs) are small non-coding RNAs which are involved in the regulation of virtually every cellular process. They regulate and control the levels of mRNA stability and post-transcriptional gene expression. Aberrant expression of miRNAs has been associated in many disease states, and miRNA-based therapies are in progress. In this study, we investigated possible variations of miRNA expression profiles of respiratory epithelial cells infected by invasive Candida species. For this purpose, respiratory epithelial tissues of infected individuals from hospital laboratory were accessed before their treatment. Invasive Candida infection was confirmed by isolation of Candia albicans from the blood cultures of the same infected individuals. The purity of epithelial tissues was assessed by flow cytometry (FACSCalibur cytometer; BD Biosciences, Heidelberg, Germany) using statin antibody (S-44). TaqMan quantitative real-time PCR (in a TaqMan Low Density Array format) was used for miRNA expression profiling. MiRNAs investigated, the levels of expression of 55 miRNA were significantly altered in infected tissues. Some miRNAs showed dramatic increase (miR-16-1) or decrease of expression (miR-17-3p) as compared to control. Gene ontology enrichment analysis of these miRNA-targeted genes suggests that Candidal infection affect many important biological pathways. In summary, disturbance in miRNA expression levels indicated the change in cascade of pathological processes and the regulation of respiratory epithelial functions following invasive Candidal infection. These findings contribute to our understanding of host cell response to Candidal systemic infections.

Host genetics of invasive Aspergillus and Candida infections.

Invasive candidiasis and aspergillosis are major complications in surgical and onco-hematological patients, and still associated with an important morbidity and mortality. A large number of studies highlighted the potential role of host genetic polymorphisms that may influence susceptibility to fungal pathogens, but many were limited by insufficient statistical power, problematic design, and/or lack of replication. However, some relevant polymorphisms are now emerging from well-conducted studies whose associations have been replicated and/or are supported by strong biological evidence. Such polymorphisms together with other biomarkers may play a role in the prediction, diagnosis, and management of severe fungal infections in high-risk patients in the coming years.

Safety and pharmacokinetic profiles of repeated-dose micafungin in children and adolescents treated for invasive candidiasis.

BACKGROUND: Micafungin is an echinocandin with proven efficacy against a broad range of fungal infections, including those caused by Candida spp. OBJECTIVE: To evaluate the safety and pharmacokinetics of once-daily 3 mg/kg and 4.5 mg/kg micafungin in children with proven, probable or suspected invasive candidiasis. METHODS: Micafungin safety and pharmacokinetics were assessed in 2 phase I, open-label, repeat-dose trials. In Study 2101, children aged 2-16 years were grouped by weight to receive 3 mg/kg (≥25 kg) or 4.5 mg/kg (<25 kg) intravenous micafungin for 10-14 days. In Study 2102, children aged 4 months to <2 years received 4.5 mg/kg micafungin. Study protocols were otherwise identical. RESULTS: Safety was analyzed in 78 and 9 children in Studies 2101 and 2102, respectively. Although adverse events (AEs) were experienced by most children (2101: n=62; 2102: n=9), micafungin-related AEs were less common (2101: n=28; 2102: n=1), and the number of patients discontinuing due to AEs was low (2101: n=4; 2102: n=1). The most common micafungin-related AEs were infusion-associated symptoms, pyrexia and hypomagnesemia (Study 2101), and liver function abnormalities (Study 2102). The micafungin pharmacokinetic profile was similar to that seen in other studies conducted in children, but different than that observed in adults. CONCLUSIONS: In this small cohort of children, once-daily doses of 3 mg/kg and 4.5 mg/kg micafungin were well tolerated. Pharmacokinetic data will be combined in a population pharmacokinetic analysis to support US dosing recommendations in children.

Efficacy and toxicity of a tropically stable lipid-based formulation of amphotericin B (iCo-010) in a rat model of invasive candidiasis.

The objective of this work was to assess the antifungal activity of a tropically stable formulation of amphotericin B (AmB) (iCo-010) over short period of treatment in a rat model of invasive candidiasis. The rats were infected with Candida albicans (ATCC 18804); 48 h later, the animals were assigned either to a control group, AmBisome(®) group (5 mg/kg QD), or iCo-010 groups (0.5, 1, 2.5, 5 and 10 mg/kg TID). The animals were treated for two days and then sacrificed 18 h following the completion of the treatment. The blood, liver, lungs, kidneys and spleen were harvested to assess the colony forming units in the samples. There was no significant difference in the reduction of the fungal burden in the organs between the AmBisome(®) and iCo-010 groups except in the spleen and liver. There was a linear correlation between the antifungal activity in renal tissues and the administered doses of iCo-010. The plasma creatinine levels were not significantly different among the control and all the treatment groups. Oral iCo-010 has high efficacy against invasive candidiasis in renal and pulmonary tissues. Longer treatment period than the two-days regimen should be considered for higher therapeutic efficacy of iCo-010 in all the tissues.

Echinocandins: addressing outstanding questions surrounding treatment of invasive fungal infections.

PURPOSE: Recent in vitro and clinical data addressing outstanding issues regarding the selection, dosing, and monitoring of echinocandins for the treatment of invasive fungal infections (IFIs) are reviewed. SUMMARY: The echinocandins (caspofungin, micafungin, and anidulafungin) are attractive treatment options for the treatment of select IFIs, most notably invasive candidiasis and treatment-refractory invasive aspergillosis. A literature review of English-language articles published between January 2007 and May 2010 was performed using the terms caspofungin, micafungin, anidulafungin, and echinocandin. In vitro, in vivo, and both pediatric and adult clinical studies and case reports were included. The challenges to establish meaningful interpretive criteria for in vitro testing of yeasts continue to persist, as do the establishment of the clinical relevancy of both the reduced in vitro susceptibilities to Candida parapsilosis and the paradoxical growth of Candida species at higher dosages. Despite increasing use of these agents and reports of breakthrough infections, echinocandins have continued to maintain potency against a broad spectrum of Candida and Aspergillus species. Recent in vitro studies also support the excellent activity of echinocandins against Candida biofilms. While recent published studies have better defined dosing in special populations (such as pediatric patients and those with organ dysfunction), attempts to increase efficacy by dosage intensification have been unsuccessful. Several pharmacoeconomic studies have been performed in attempts to justify the high acquisition costs of these drugs. In general, these studies found that echinocandins may be cost-effective for such indications. CONCLUSION: Available in vitro data, animal studies, and clinical studies do not clearly differentiate agents in the echinocandin class. Clinical data continue to support the use of echinocandins as a safe and well-tolerated treatment option for candidemia and invasive aspergillosis.

Safety and pharmacokinetics of multiple-dose anidulafungin in infants and neonates.

Candida infections are common and often fatal in infants and neonates. Anidulafungin has excellent activity against Candida species, but the pharmacokinetics (PK) and safety of the drug in infants and neonates are unknown. The object of our study was to determine the PK and safety of anidulafungin in infants and neonates at risk for invasive candidiasis. Intravenous anidulafungin (1.5 mg/kg/day maintenance dose) was administered to 15 infants and neonates over 3 to 5 days. Plasma samples were collected after the first dose and again after the third to fifth doses. The pharmacokinetic parameters of the drug were determined by noncompartmental analysis. Safety was assessed using National Cancer Institute common toxicity criteria. The study showed that drug exposure levels were similar between neonates and infants; the median areas under the concentration-time curve (range) was 75 (30-109) µg·h/ml and 98 (55-278) µg·h/ml (P = 0.12) for neonates and infants, respectively. No drug-related serious adverse events were observed. The study results indicate that neonates and infants receiving 1.5 mg/kg/day have anidulafungin exposure levels similar to those in children receiving similar weight-based dosing and in adult patients receiving 100 mg/day.