Crosstalk between the calcineurin and cell wall integrity pathways prevents chitin overexpression in Candida albicans.

Echinocandins such as caspofungin are frontline antifungal drugs that compromise ß-1,3 glucan synthesis in the cell wall. Recent reports have shown that fungal cells can resist killing by caspofungin by upregulation of chitin synthesis, thereby sustaining cell wall integrity (CWI). When echinocandins are removed, the chitin content of cells quickly returns to basal levels, suggesting that there is a fitness cost associated with having elevated levels of chitin in the cell wall. We show here that simultaneous activation of the calcineurin and CWI pathways generates a subpopulation of Candida albicans yeast cells that have supra-normal chitin levels interspersed throughout the inner and outer cell wall, and that these cells are non-viable, perhaps due to loss of wall elasticity required for cell expansion and growth. Mutations in the Ca2+-calcineurin pathway prevented the formation of these non-viable supra-high chitin cells by negatively regulating chitin synthesis driven by the CWI pathway. The Ca2+-calcineurin pathway may therefore act as an attenuator that prevents the overproduction of chitin by coordinating both chitin upregulation and negative regulation of the CWI signaling pathway. This article has an associated First Person interview with the first author of the paper.

Monoclonal Antibodies Targeting Surface-Exposed Epitopes of Candida albicans Cell Wall Proteins Confer In Vivo Protection in an Infection Model.

Monoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

Targeted changes of the cell wall proteome influence Candida albicans ability to form single- and multi-strain biofilms.

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.

The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis.

Destruction of the pulmonary epithelium is a major feature of lung diseases caused by the mould pathogen Aspergillus fumigatus. Although it is widely postulated that tissue invasion is governed by fungal proteases, A. fumigatus mutants lacking individual or multiple enzymes remain fully invasive, suggesting a concomitant requirement for other pathogenic activities during host invasion. In this study we discovered, and exploited, a novel, tissue non-invasive, phenotype in A. fumigatus mutants lacking the pH-responsive transcription factor PacC. Our study revealed a novel mode of epithelial entry, occurring in a cell wall-dependent manner prior to protease production, and via the Dectin-1 ß-glucan receptor. ΔpacC mutants are defective in both contact-mediated epithelial entry and protease expression, and significantly attenuated for pathogenicity in leukopenic mice. We combined murine infection modelling, in vivo transcriptomics, and in vitro infections of human alveolar epithelia, to delineate two major, and sequentially acting, PacC-dependent processes impacting epithelial integrity in vitro and tissue invasion in the whole animal. We demonstrate that A. fumigatus spores and germlings are internalised by epithelial cells in a contact-, actin-, cell wall- and Dectin-1 dependent manner and ΔpacC mutants, which aberrantly remodel the cell wall during germinative growth, are unable to gain entry into epithelial cells, both in vitro and in vivo. We further show that PacC acts as a global transcriptional regulator of secreted molecules during growth in the leukopenic mammalian lung, and profile the full cohort of secreted gene products expressed during invasive infection. Our study reveals a combinatorial mode of tissue entry dependent upon sequential, and mechanistically distinct, perturbations of the pulmonary epithelium and demonstrates, for the first time a protective role for Dectin-1 blockade in epithelial defences. Infecting ΔpacC mutants are hypersensitive to cell wall-active antifungal agents highlighting the value of PacC signalling as a target for antifungal therapy.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

Caspofungin Treatment of Aspergillus fumigatus Results in ChsG-Dependent Upregulation of Chitin Synthesis and the Formation of Chitin-Rich Microcolonies.

Treatment of Aspergillus fumigatus with echinocandins such as caspofungin inhibits the synthesis of cell wall ß-1,3-glucan, which triggers a compensatory stimulation of chitin synthesis. Activation of chitin synthesis can occur in response to sub-MICs of caspofungin and to CaCl2 and calcofluor white (CFW), agonists of the protein kinase C (PKC), and Ca(2+)-calcineurin signaling pathways. A. fumigatus mutants with the chs gene (encoding chitin synthase) deleted (ΔAfchs) were tested for their response to these agonists to determine the chitin synthase enzymes that were required for the compensatory upregulation of chitin synthesis. Only the ΔAfchsG mutant was hypersensitive to caspofungin, and all other ΔAfchs mutants tested remained capable of increasing their chitin content in response to treatment with CaCl2 and CFW and caspofungin. The resulting increase in cell wall chitin content correlated with reduced susceptibility to caspofungin in the wild type and all ΔAfchs mutants tested, with the exception of the ΔAfchsG mutant, which remained sensitive to caspofungin. In vitro exposure to the chitin synthase inhibitor, nikkomycin Z, along with caspofungin demonstrated synergistic efficacy that was again AfChsG dependent. Dynamic imaging using microfluidic perfusion chambers demonstrated that treatment with sub-MIC caspofungin resulted initially in hyphal tip lysis. However, thickened hyphae emerged that formed aberrant microcolonies in the continued presence of caspofungin. In addition, intrahyphal hyphae were formed in response to echinocandin treatment. These in vitro data demonstrate that A. fumigatus has the potential to survive echinocandin treatment in vivo by AfChsG-dependent upregulation of chitin synthesis. Chitin-rich cells may, therefore, persist in human tissues and act as the focus for breakthrough infections.

Cell wall stress induces alternative fungal cytokinesis and septation strategies.

In fungi, as with all walled organisms, cytokinesis followed by septation marks the end of the cell cycle and is essential for cell division and viability. For yeasts, the septal cross-wall comprises a ring and primary septal plate composed of chitin, and a secondary septum thickened with ß(1,3)-glucan. In the human pathogen Candida albicans, chitin synthase enzyme Chs1 builds the primary septum that is surrounded by a chitin ring made by Chs3. Here we show that the lethal phenotype induced by repression of CHS1 was abrogated by stress-induced synthesis of alternative and novel septal types synthesized by other chitin synthase enzymes that have never before been implicated in septation. Chs2 and Chs8 formed a functional salvage septum, even in the absence of both Chs1 and Chs3. A second type of salvage septum formed by Chs2 in combination with Chs3 or Chs8 was proximally offset in the mother-bud neck. Chs3 alone or in combination with Chs8 formed a greatly thickened third type of salvage septum. Therefore, cell wall stress induced alternative forms of septation that rescued cell division in the absence of Chs1, demonstrating that fungi have previously unsuspected redundant strategies to enable septation and cell division to be maintained, even under potentially lethal environmental conditions.

Differential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1.

The ß-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.

Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors.

The present work reports the effects of caspofungin, a ß-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ß-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ß-glucan synthase inhibitor against this fungus.

Elevated chitin content reduces the susceptibility of Candida species to caspofungin.

The echinocandin antifungal drugs inhibit synthesis of the major fungal cell wall polysaccharide ß(1,3)-glucan. Echinocandins have good efficacy against Candida albicans but reduced activity against other Candida species, in particular Candida parapsilosis and Candida guilliermondii. Treatment of Candida albicans with a sub-MIC level of caspofungin has been reported to cause a compensatory increase in chitin content and to select for sporadic echinocandin-resistant FKS1 point mutants that also have elevated cell wall chitin. Here we show that elevated chitin in response to caspofungin is a common response in various Candida species. Activation of chitin synthesis was observed in isolates of C. albicans, Candida tropicalis, C. parapsilosis, and C. guilliermondii and in some isolates of Candida krusei in response to caspofungin treatment. However, Candida glabrata isolates demonstrated no exposure-induced change in chitin content. Furthermore, isolates of C. albicans, C. krusei, C. parapsilosis, and C. guilliermondii which were stimulated to have higher chitin levels via activation of the calcineurin and protein kinase C (PKC) signaling pathways had reduced susceptibility to caspofungin. Isolates containing point mutations in the FKS1 gene generally had higher chitin levels and did not demonstrate a further compensatory increase in chitin content in response to caspofungin treatment. These results highlight the potential of increased chitin synthesis as a potential mechanism of tolerance to caspofungin for the major pathogenic Candida species.

Carbon source-induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans.

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose-grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate-grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose-grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate-grown cells. We identified mating and pheromone-regulated proteins that were exclusive to lactate-grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa-specific and other niche-specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.

Elevated cell wall chitin in Candida albicans confers echinocandin resistance in vivo.

Candida albicans cells with increased cell wall chitin have reduced echinocandin susceptibility in vitro. The aim of this study was to investigate whether C. albicans cells with elevated chitin levels have reduced echinocandin susceptibility in vivo. BALB/c mice were infected with C. albicans cells with normal chitin levels and compared to mice infected with high-chitin cells. Caspofungin therapy was initiated at 24 h postinfection. Mice infected with chitin-normal cells were successfully treated with caspofungin, as indicated by reduced kidney fungal burdens, reduced weight loss, and decreased C. albicans density in kidney lesions. In contrast, mice infected with high-chitin C. albicans cells were less susceptible to caspofungin, as they had higher kidney fungal burdens and greater weight loss during early infection. Cells recovered from mouse kidneys at 24 h postinfection with high-chitin cells had 1.6-fold higher chitin levels than cells from mice infected with chitin-normal cells and maintained a significantly reduced susceptibility to caspofungin when tested in vitro. At 48 h postinfection, caspofungin treatment induced a further increase in chitin content of C. albicans cells harvested from kidneys compared to saline treatment. Some of the recovered clones had acquired, at a low frequency, a point mutation in FKS1 resulting in a S645Y amino acid substitution, a mutation known to confer echinocandin resistance. This occurred even in cells that had not been exposed to caspofungin. Our results suggest that the efficacy of caspofungin against C. albicans was reduced in vivo due to either elevation of chitin levels in the cell wall or acquisition of FKS1 point mutations.

High-Pressure Freezing and Transmission Electron Microscopy to Visualize the Ultrastructure of the C. auris Cell Wall.

Transmission electron microscopy (TEM) is the main technique used to study the ultrastructure of biological samples. Chemical fixation was considered the main method for preserving samples for TEM; however, it is a relatively slow method of fixation and can result in morphological alterations. Cryofixation using high-pressure freezing (HPF) overcomes the limitations of chemical fixation by preserving samples instantly. Here, we describe our HPF methods optimized for visualizing Candida auris at the ultrastructural level.

Fungal echinocandin resistance.

The echinocandins are the newest class of antifungal agents in the clinical armory. These secondary metabolites are non-competitive inhibitors of the synthesis of beta-(1,3)-glucan, a major structural component of the fungal cell wall. Recent work has shown that spontaneous mutations can arise in two hot spot regions of Fks1 the target protein of echinocandins that reduce the enzyme's sensitivity to the drug. However, other strains have been isolated in which the sequence of FKS1 is unaltered yet the fungus has decreased sensitivity to echinocandins. In addition it has been shown that echinocandin-treatment can induce cell wall salvage mechanisms that result in the compensatory upregulation of chitin synthesis in the cell wall. This salvage mechanism strengthens cell walls damaged by exposure to echinocandins. Therefore, fungal resistance to echinocandins can arise due to the selection of either stable mutational or reversible physiological alterations that decrease susceptibility to these antifungal agents.

Stimulation of chitin synthesis rescues Candida albicans from echinocandins.

Echinocandins are a new generation of novel antifungal agent that inhibit cell wall beta(1,3)-glucan synthesis and are normally cidal for the human pathogen Candida albicans. Treatment of C. albicans with low levels of echinocandins stimulated chitin synthase (CHS) gene expression, increased Chs activity, elevated chitin content and reduced efficacy of these drugs. Elevation of chitin synthesis was mediated via the PKC, HOG, and Ca(2+)-calcineurin signalling pathways. Stimulation of Chs2p and Chs8p by activators of these pathways enabled cells to survive otherwise lethal concentrations of echinocandins, even in the absence of Chs3p and the normally essential Chs1p, which synthesize the chitinous septal ring and primary septum of the fungus. Under such conditions, a novel proximally offset septum was synthesized that restored the capacity for cell division, sustained the viability of the cell, and abrogated morphological and growth defects associated with echinocandin treatment and the chs mutations. These findings anticipate potential resistance mechanisms to echinocandins. However, echinocandins and chitin synthase inhibitors synergized strongly, highlighting the potential for combination therapies with greatly enhanced cidal activity.

Caspofungin Induced Cell Wall Changes of Candida Species Influences Macrophage Interactions.

Candida species are known to differ in their ability to cause infection and have been shown to display varied susceptibilities to antifungal drugs. Treatment with the echinocandin, caspofungin, leads to compensatory alterations in the fungal cell wall. This study was performed to compare the structure and composition of the cell walls of different Candida species alone and in response to caspofungin treatment, and to evaluate how changes at the fungal cell surface affects interactions with macrophages. We demonstrated that the length of the outer fibrillar layer varied between Candida species and that, in most cases, reduced fibril length correlated with increased exposure of ß-1,3-glucan on the cell surface. Candida glabrata and Candida guilliermondii, which had naturally more ß-1,3-glucan exposed on the cell surface, were phagocytosed significantly more efficiently by J774 macrophages. Treatment with caspofungin resulted in increased exposure of chitin and ß-1,3-glucan on the surface of the majority of Candida species isolates that were tested, with the exception of C. glabrata and Candida parapsilosis isolates. This increase in exposure of the inner cell wall polysaccharides, in most cases, correlated with reduced uptake by macrophages and in turn, a decrease in production of TNFα. Here we show that differences in the exposure of cell wall carbohydrates and variations in the repertoire of covalently attached surface proteins of different Candida species contributes to their recognition by immune cells.

Erratum to "Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation" [Cell Surf. 1 (2018) 43-56].

[This corrects the article DOI: 10.1016/j.tcsw.2018.03.002.].