Malassezia sympodialis Mala s 1 allergen is a potential KELCH protein that cross reacts with human skin.

Malassezia are the dominant commensal yeast species of the human skin microbiota and are associated with inflammatory skin diseases, such as atopic eczema (AE). The Mala s 1 allergen of Malassezia sympodialis is a ß-propeller protein, inducing both IgE and T-cell reactivity in AE patients. We demonstrate by immuno-electron microscopy that Mala s 1 is mainly located in the M. sympodialis yeast cell wall. An anti-Mala s 1 antibody did not inhibit M. sympodialis growth suggesting Mala s 1 may not be an antifungal target. In silico analysis of the predicted Mala s 1 protein sequence identified a motif indicative of a KELCH protein, a subgroup of ß-propeller proteins. To test the hypothesis that antibodies against Mala s 1 cross-react with human skin (KELCH) proteins we examined the binding of the anti-Mala s 1 antibody to human skin explants and visualized binding in the epidermal skin layer. Putative human targets recognized by the anti-Mala s 1 antibody were identified by immunoblotting and proteomics. We propose that Mala s 1 is a KELCH-like ß-propeller protein with similarity to human skin proteins. Mala s 1 recognition may trigger cross-reactive responses that contribute to skin diseases associated with M. sympodialis.

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.

Neutralisation of SARS-CoV-2 by anatomical embalming solutions.

Teaching and learning anatomy by using human cadaveric specimens has been a foundation of medical and biomedical teaching for hundreds of years. Therefore, the majority of institutions that teach topographical anatomy rely on body donation programmes to provide specimens for both undergraduate and postgraduate teaching of gross anatomy. The COVID-19 pandemic has posed an unprecedented challenge to anatomy teaching because of the suspension of donor acceptance at most institutions. This was largely due to concerns about the potential transmissibility of the SARS-CoV-2 virus and the absence of data about the ability of embalming solutions to neutralise the virus. Twenty embalming solutions commonly used in institutions in the United Kingdom and Ireland were tested for their ability to neutralise SARS-CoV-2, using an established cytotoxicity assay. All embalming solutions tested neutralised SARS-CoV-2, with the majority of solutions being effective at high-working dilutions. These results suggest that successful embalming with the tested solutions can neutralise the SARS-CoV-2 virus, thereby facilitating the safe resumption of body donation programmes and cadaveric anatomy teaching.

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.

The potential of respiration inhibition as a new approach to combat human fungal pathogens.

The respiratory chain has been proposed as an attractive target for the development of new therapies to tackle human fungal pathogens. This arises from the presence of fungal-specific electron transport chain components and links between respiration and the control of virulence traits in several pathogenic species. However, as the physiological roles of mitochondria remain largely undetermined with respect to pathogenesis, its value as a potential new drug target remains to be determined. The use of respiration inhibitors as fungicides is well developed but has been hampered by the emergence of rapid resistance to current inhibitors. In addition, recent data suggest that adaptation of the human fungal pathogen, Candida albicans, to respiration inhibitors can enhance virulence traits such as yeast-to-hypha transition and cell wall organisation. We conclude that although respiration holds promise as a target for the development of new therapies to treat human fungal infections, we require a more detailed understanding of the role that mitochondria play in stress adaption and virulence.

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron.

Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Sfp1 and Rtg3 reciprocally modulate carbon source-conditional stress adaptation in the pathogenic yeast Candida albicans.

The pathogenicity of the clinically important yeast, Candida albicans, is dependent on robust responses to host-imposed stresses. These stress responses have generally been dissected in vitro at 30°C on artificial growth media that do not mimic host niches. Yet host inputs, such as changes in carbon source or temperature, are known to affect C. albicans stress adaptation. Therefore, we performed screens to identify novel regulators that promote stress resistance during growth on a physiologically relevant carboxylic acid and at elevated temperatures. These screens revealed that, under these 'non-standard' growth conditions, numerous uncharacterised regulators are required for stress resistance in addition to the classical Hog1, Cap1 and Cta4 stress pathways. In particular, two transcription factors (Sfp1 and Rtg3) promote stress resistance in a reciprocal, carbon source-conditional manner. SFP1 is induced in stressed glucose-grown cells, whereas RTG3 is upregulated in stressed lactate-grown cells. Rtg3 and Sfp1 regulate the expression of key stress genes such as CTA4, CAP1 and HOG1 in a carbon source-dependent manner. These mechanisms underlie the stress sensitivity of C. albicans sfp1 cells during growth on glucose, and rtg3 cells on lactate. The data suggest that C. albicans exploits environmentally contingent regulatory mechanisms to retain stress resistance during host colonisation.

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.

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

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.

Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation.

BACKGROUND: Biofilm formation by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity. RESULTS: Initially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24 h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (∆cht2, ∆cht3) and double knockout (∆cht2/∆cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold. CONCLUSIONS: Collectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity.