Endosomal Trafficking as a Determinant of Antifungal Tolerance in the Pathogenic Fungus Candida albicans

Several important antifungal drugs inhibit the synthesis of ergosterol, a lipid that modulates the thickness, fluidity and permeability of fungal cell membranes. These include the azole antifungals, which block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of ‘toxic’ sterol intermediates are both thought to cause plasma membrane dysfunction and ultimately growth arrest. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole, and determine if, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole was observed as an early consequence of azole treatment, before significant growth inhibition was observed. Using a genetic approach, we determined that vacuole fragmentation was a consequence of Erg11p inhibition rather than an off-target effect of azole treatment. Moreover, the degree of vacuole fragmentation following azole treatment was influenced by Erg3p, an enzyme in the ergosterol biosynthetic pathway involved in the production of toxic sterol intermediaries upon Erg11p inhibition. We also determined that vacuolar trafficking significantly impacts C. albicans susceptibility to azole antifungals and other ergosterol biosynthesis inhibitors. For instance, a vps21∆/∆ mutant, blocked in membrane trafficking through the pre-vacuolar compartment (PVC), grew significantly more than wild-type controls in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21∆/∆ mutant was able to grow in the presence of the azoles despite depletion of cellular ergosterol. This phenotype resembles an exaggerated form of azole tolerance known as ‘trailing growth’, which has been described for some clinical isolates. In contrast, the vps21∆/∆ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. The azole tolerance phenotype of the C. albicans vps21Δ/Δ mutant was independent of known azole resistance mechanisms such as the efflux pumps Cdr1p and Mdr1p. Moreover, the azole tolerance of the vps21Δ/Δ mutant was influenced by both pH and incubation temperature, consistent with trailing growth phenotypes. The C. albicans vps21Δ/Δ mutant exhibits less plasma membrane permeabilization upon azole treatment, as determined by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has elevated levels of intracellular calcium and enhanced calcineurin activity, as evidenced by increased expression of a calcineurin responsive RTA2-GFP reporter construct in response to fluconazole. Furthermore, the azole tolerant phenotype of the vps21Δ/Δ mutant is dependent upon both calcium and calcineurin signaling. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment through modulation of intracellular calcium levels and calcineurin dependent responses. While we determined that deletion of VPS21 alone was not sufficient to confer a survival advantage upon C. albicans following azole treatment in the mouse model of vaginal candidiasis, the azole susceptibility of the vps21Δ/Δ mutant in the mouse model disseminated infection is yet to be tested. Moreover, it is unclear how, or if the azole tolerant phenotype of the vps21Δ/Δ mutant relates to that of trailing clinical isolates, or if these clinical isolates have abnormal endosomal trafficking

Species-specific differences in C-5 sterol desaturase function influence the outcome of azole antifungal exposure

The azole antifungals inhibit sterol 14α-demethylase (S14DM), leading to depletion of cellular ergosterol and the synthesis of an aberrant sterol diol that disrupts membrane function. In Candida albicans, sterol diol production is catalyzed by the C-5 sterol desaturase enzyme encoded by ERG3. Accordingly, mutations that inactivate ERG3 enable the fungus to grow in the presence of the azoles. The purpose of this study was to compare the propensities of C-5 sterol desaturases from different fungal pathogens to produce the toxic diol upon S14DM inhibition and thus contribute to antifungal efficacy. The coding sequences of ERG3 homologs from C. albicans (CaERG3), Candida glabrata (CgERG3), Candida auris (CaurERG3), Cryptococcus neoformans (CnERG3), Aspergillus fumigatus (AfERG3A-C) and Rhizopus delemar (RdERG3A/B) were expressed in a C. albicans erg3Δ/Δ mutant to facilitate comparative analysis. All but one of the Erg3p-like proteins (AfErg3C) at least partially restored C-5 sterol desaturase activity and to corresponding degrees rescued the stress and hyphal growth defects of the C. albicans erg3Δ/Δ mutant, confirming functional equivalence. Each C-5 desaturase enzyme conferred markedly different responses to fluconazole exposure in terms of the MIC and residual growth observed at supra-MICs. Upon fluconazole-mediated inhibition of S14DM, the strains expressing each homolog also produced various levels of 14α-methylergosta-8,24(28)-dien-3β,6α-diol. The RdErg3A and AfErg3A proteins are notable for low levels of sterol diol production and failing to confer appreciable azole sensitivity upon the C. albicans erg3Δ/Δ mutant. These findings suggest that species-specific properties of C-5 sterol desaturase may be an important determinant of intrinsic azole sensitivity

Methodologies for in vitro and in vivo evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms

KT acknowledges receipt of a mandate of Industrial Research Fund (IOFm/05/022). JB acknowledges funding from the European Research Council Advanced Award 3400867/RAPLODAPT and the Israel Science Foundation grant # 314/13 (www.isf.il). NG acknowledges the Wellcome Trust and MRC for funding. CD acknowledges funding from the Agence Nationale de Recherche (ANR-10-LABX-62-IBEID). CJN acknowledges funding from the National Institutes of Health R35GM124594 and R21AI125801. AW is supported by the Wellcome Trust Strategic Award (grant 097377), the MRC Centre for Medical Mycology (grant MR/N006364/1) at the University of Aberdeen MaCA: outside this study MaCA has received personal speaker’s honoraria the past five years from Astellas, Basilea, Gilead, MSD, Pfizer, T2Candida, and Novartis. She has received research grants and contract work paid to the Statens Serum Institute from Astellas, Basilea, Gilead, MSD, NovaBiotics, Pfizer, T2Biosystems, F2G, Cidara, and Amplyx. CAM acknowledges the Wellcome Trust and the MRC MR/N006364/1. PVD, TC and KT acknowledge the FWO research community: Biology and ecology of bacterial and fungal biofilms in humans (FWO WO.009.16N). AAB acknowledges the Deutsche Forschungsgemeinschaft – CRC FungiNet.Peer reviewedPublisher PD

Methodologies for <i>in vitro</i> and <i>in vivo</i> evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the &lt;i&gt;in vivo&lt;/i&gt; efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt; performance of anti-infective coatings and materials to prevent fungal biofilm-based infections

Advances in Antifungal Development: Discovery of New Drugs and Drug Repurposing

This reprint describes recent advances made in the field of antifungal development, especially the discovery of new drugs and drug repurposing. The articles presented in this book provide useful information and insight for the development of new antifungal drugs or intervention strategies. The identification of new, safe molecules, and cellular targets, as well as the elucidation of their antifungal mechanisms of action, will further the effective control of fungal pathogens, especially those resistant to current therapeutic agents

Drug resistance and virulence of the human fungal pathogen Candida glabrata

Candida glabrata ist ein opportunistischer humanpathogener Pilz. Nach Candida albicans stellt er die zweithäufigste Ursache für Pilzerkrankungen durch Candida spp. dar. Die Infektion mit C. glabrata kann zu Erkrankungen der Haut oder der Schleimhäute bis hin zur lebensbedrohlichen systemischen Infektion bei immunsupprimierten Patienten führen. Hauptvirulenzfaktoren von C. glabrata sind sowohl eine erhöhte natürliche Resistenz gegen Azolverbindungen, als auch eine große Anzahl verschiedener Adhesine. Welche molekularen Mechanismen diesen Virulenzfaktoren zu Grunde liegen, ist größtenteils noch unbekannt. Genomik und phänotypische Analyse von Deletionsmutanten stellen einen Ansatz dar, um neue Virulenzfaktoren pathogener Pilzen zu identifizieren. Für diese Doktorarbeit wurden C. glabrata Sequenzdaten verwendet, um nicht essentielle, zu Saccharomyces cerevisiae homologe Gene auszuwählen. Mit Hilfe eines revers-genetischen Ansatzes wurde dann eine Kollektion von Deletionsmutanten erstellt. Diese Stammbibliothek diente als Ausgangsbasis für die phänotypische Analyse der Mutanten zur Identifizierung neuer Gene, die die Pathogenität und Azolresistenz von C. glabrata beeinflussen. Insgesamt umfasst die Kollektion 476 einzelne, mit einem molekularen Barcode versehene Stämme, bei denen Signaltransduktionsgene, Transkriptionsfaktoren, Zellwandbiosynthesegene, Resistenzgene und C. glabrata spezifische Gene deletiert wurden. Es wurden 103 Mutanten identifiziert, die Wachstumsdefekte unter Zellwandstress, in Gegenwart von Antimykotika, bei Temperaturstress, Osmolaritätsänderungen, Kontakt mit Detergenzien oder beim Wachstum auf Minimalmedium aufzeigen. Die Interaktion zwischen Makrophagen und C. glabrata wurde in vitro durch die Detektion von reaktiven Sauerstoffmolekülen (ROS) getestet. Dadurch wurden mehrere Zellwandmutanten entdeckt, die eine erhöhte ROS-Produktion durch die Makrophagen verursachen. Mehrere Deletionsstämme zeigten Phänotypen, die sich von den bekannten S. cerevisiae Mutanten unterscheiden. Es konnten Gene identifiziert werden, die noch nicht mit Caspofunginsensitivität assoziiert worden sind. Darunter befanden sich auch Gene, die keine Orthologe in S. cerevisiae haben. CgCBK1 wurde aufgrund des schweren Zellteilungsdefekts näher charakterisiert. Die Transkriptionsanalyse des Cgcbk1Δ Stammes zeigte, dass bestimmte Zellwandgene unterschiedlich exprimiert werden. Diese Sammlung von Deletionsstämmen ist eine der größten weltweit und ist somit von sehr großem Nutzen für das Studium der Pathogenität von C. glabrata. Zukünftige in vivo Experimente werden unter Ausnützung der integrierten „molekularen Barcodes“ die Identifizierung neuer Virulenzfaktoren ermöglichen.Candida glabrata is an opportunistic human fungal pathogen. It is the second most frequent cause of Candida-derived infections after Candida albicans. Infection with either one of the two pathogenic fungi can result in diseases ranging from superficial cutaneous or mucosal to life-threatening systemic infections in immunocompromised individuals. Welldocumented virulence attributes of C. glabrata are the inherent reduced azole susceptibility and a large repertoire of adhesin genes, which are regulated by transcriptional silencing. However, the molecular basis of C. glabrata antifungal drug resistance and additional virulence factors is not well understood. The combination of fungal genomics and large-scale phenotypic profiling of deletion mutants represents a powerful approach to identify new factors contributing to fungal virulence. For this doctoral thesis, the C. glabrata genome sequence data were used to select genes with non-essential functional orthologues of the non-pathogenic yeast Saccharomyces cerevisiae. Based on this selection, a large-scale reverse genetics approach was initiated to identify novel genes implicated in C. glabrata pathogenicity and drug resistance. A bar-coded C. glabrata deletion strain collection was engineered comprising some 500 single gene deletion mutants affected in signaling functions, regulation of gene expression, cell wall biogenesis, transport processes, drug resistance, stress response and metabolism. Phenotypic profiling identified a total of 103 C. glabrata genes involved in resistance to cell wall-perturbing compounds, antifungal drugs, heat stress, osmosensitivity, metal ion or detergent tolerance, growth on minimal medium and phenotypic switching. Host-pathogen interaction related phenotypes were analyzed using an in vitro assay detecting reactive oxygen species (ROS). Screening for ROS elicited by primary mouse bone-marrow-derived macrophages co-incubated with C. glabrata mutants resulted in the identification of mutants lacking cell wall-related genes. Taken together, numerous deletion strains showed growth phenotypes different from known phenotypes of S. cerevisiae. For example, genes were identified, which have previously not been associated with sensitivity to the glucan synthase inhibitor Caspofungin. This also included C. glabrata genes, which do not have orthologues in baker’s yeast. The function of CgCBK1 gene has been studied in more detail, because it exhibits severe cell separation defects. Transcriptional profiling of this mutant showed differential expression of a distinct set of genes with cell wall-associated functions. In summary, the generated C. glabrata gene deletion strain collection is one of the largest collections of fungal deletion strains in the world and represents a powerful tool to study the virulence of a human fungal pathogen. Future studies based on the in vitro results will exploit the signature-tag strategy to identify novel virulence-associated factors in vivo

Feeling the Heat: Investigating the dual assault of Zymoseptoria tritici and Heat Stress on Wheat (Triticum aestivum)

As a result of climate change, field conditions are increasingly challenging for crops. Research has shown how elevated temperatures affect crop performance, yet the impact of temperature on host-pathogen relationships remains unknown. Understanding the effects of combined abiotic and biotic stresses on crop plants and the plant-microbial interaction is crucial in developing strategies to improve crop stress tolerance and manage diseases effectively. Lipids sense, signal, and mitigate temperature elevation effects, and lipid remodelling plays a key role in the plant and fungal response to heat stress. Our study uses a systems approach to examine the Z. tritici wheat model system, combining transcriptomics, lipidomics, and phenotyping to decipher the impact of high-temperature stress on the plant-pathogen interaction. Microscopy in vivo and RNA-Seq analyses confirmed that Z. tritici responds to high-temperature treatments with morphological and transcriptomic changes. Temperature-related configuration of the transcriptome was associated with the accessory chromosomes and expression of ‘accessory’ pan-genome-derived genes. Metabolism-related gene expression predominated, indicated by GO enrichment and analysis of KOG classes, and large-scale lipid remodelling was likely given the proportion of lipid transport and metabolism-related expression changes in response to temperature. Changes in lipid content and composition were then validated by LC-MS analysis. Heat-responsive fungal genes and pathways, including scramblase family genes, are being tested by reverse genetics to ascertain their importance for fungal adaption to elevated temperatures. Elevated temperature schemes were applied to wheat to study the impact of combined stress on the plant-pathogen interaction, based on long-term climate data from Rothamsted Research, using transcriptomic, lipidomic and phenotypic analyses. Comparing non-infected and infected wheat plants under typical and elevated temperatures. Our initial analysis of the transcriptomic data indicates a delay in the development of Z. tritici, followed by its adaptation to the warmer environment. Once the infection was established, the fungus exhibited resilience to the impact of higher external temperatures. Our results indicate that temperature elevations associated with climate change directly impact plant-pathogen interactions. Furthermore, the study demonstrates a need for further detailed understanding to sustain crop resilience

Comparative genomics and systems biology of environmental stress responses relevant to fungal virulence

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Candida spp. and Oxidative Stress Response in Innate Immune Cells

In dieser Arbeit wurde der „Oxidative Burst“ des angeborenen Immunsystems in der Interaktion mit Candida albicans untersucht. Das klinische Spektrum des opportunistischen Pathogens C. albicans reicht von mucokutanen Infektionen bis hin zu lebensbedrohlichen, systemischen Krankheiten in immunsupprimierten Patienten. Eine der ersten Reaktionen der Zellen des angeborenen Immunsystems, sogenannte Phagozyten, ist die Produktion von Reaktiven Oxygen Spezies (ROS) wenn sie auf Pathogene stoßen. ROS spielen eine wichtige Rolle bei Entzündungsreaktionen, zum Beispiel zerstören sie eindringende Krankheitserreger. Durch eine Überproduktion von ROS kann aber auch das Endothel beschädigt werden. Frühere Studien haben gezeigt, dass Zymosan, eine Zellwand Aufbereitung von Saccharomyces cerevisiae, und C. albicans die ROS Produktion in Makrophagen aktivieren. Das C. albicans Genom codiert sechs Superoxid Dismutasen (SOD1 bis SOD6), die an der Zersetzung von ROS beteiligt sind, SOD1 bis SOD3 sind intrazellular und SOD4 bis SOD6 sind wahrscheinlich an der Zellwand von C. albicans lokalisiert. Diese Arbeit zeigt, dass die Co-Kultur von Makrophagen oder myeloischen dendritischen Zellen mit C. albicans denen Sod5 genetisch entfernt wurde zu einer massiven extrazellulären Anhäufung von ROS in vitro führt. Diese ROS Akkumulierung ist in der Interaktion mit Makrophagen noch höher wenn C. albicans weder Sod4 noch Sod5 haben. Weiteres werden C. albicans Sod5 und Sod4 Mutanten von Makrophagen in vitro besser getötet als Wildtyp C. albicans. Makrophagen, die einen Defekt im Oxidativen Burst haben weil ihnen das gp91Phox Gen fehlt, können diese Mutanten nicht mehr töten, dies zeigt eine ROS-abhängige Eliminierung von pathogenen Pilzen durch Makrophagen. Diese Daten zeigen die physiologische Rolle der C. albicans Zellwand SODs bei der Entgiftung von ROS und weisen auf einen Mechanismus, mit dem C. albicans das Immunsystems in vivo überlistet, hin. Im zweiten Teil dieser Arbeit wurden potentielle Rezeptor(en) untersucht, durch die Makrophagen C. albicans erkennen, um den oxidative Burst zu induzieren. Die Toll Like Rezeptor-Familie und das intrazelluläre MyD88 Adapter-Protein sind nicht an der ROS-Produktion durch Zymosan oder C. albicans Stimulation beteiligt. Wenn der C-Typ-Lectin-Rezeptor Dectin-1 mit Zymosan oder Hitze-getöteter C. albicans stimuliert wird, induziert Dectin-1 die ROS Antwort indem die Src und Syk-Kinase aktiviert wird. Darüber hinaus aktiviert Zymosan auch die ERK1/2 MAP-Kinasen via Dectin-1. Im Gegensatz dazu ist Dectin-1 nur mäßig an der Aktivierung von ROS und ERK1 beteiligt wenn die Makrophagen mit lebenden C. albicans stimuliert werden. Interessanterweise ist die Aktivierung der Src und Syk-Kinasen auch wichtig für ROS Induktion durch Stimulierung mit lebender C. albicans. Dies führt zu dem Schluss, dass ein Rezeptor oder Adapter-Protein mit einem ITAM Motif an der Induktion von ROS beteiligt ist. Ein siRNA-basierendes knock-down-Experiment zeigt, dass das ITAM Adapter-Protein DAP12 für die ROS Produktion durch C. albicans und Zymosan mitverantwortlich ist.In this work the oxidative burst of the innate immune system in response to Candida albicans infection was investigated. The clinical spectrum of the human opportunistic pathogen C. albicans ranges from mucocutaneous infections to systemic life-threatening diseases in immunocompromised patients. One of the immediate early responses of cells of the innate immune system on encountering microbial pathogens is the production of reactive oxygen species (ROS) by phagocytes. ROS play important roles in inflammatory reactions by destroying invading pathogens. However, overproduction of ROS may also cause endothelial damage, and excessive inflammation. Previous studies have shown that zymosan, a cell wall preparation of Saccharomyces cerevisiae, as well as C. albicans in the yeast form, strongly induce ROS in macrophages. The C. albicans genome harbours six superoxide dismutases (SOD1-6) involved in ROS degradation; SOD1 to SOD3 are intracellular and SOD4 to SOD6 are located in the cell wall. This work demonstrates that co-culture of macrophages or myeloid dendritic cells with C. albicans cells lacking Sod5 leads to massive extracellular ROS accumulation in vitro. ROS accumulation was further increased in co-culture with fungal cells lacking both Sod4 and Sod5. Survival experiments show that C. albicans Sod5 and Sod4 double mutants exhibit a severe loss of viability in the presence of macrophages in vitro. The reduced viability of the mutants relative to wild type is not evident with macrophages from gp91phox-/- mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans can evade host immune surveillance in vivo. The second part of this thesis aims to identify putative receptor(s) by which macrophages recognise C. albicans and induce the oxidative burst. The Toll-like receptor family and its MyD88 adaptor protein are not involved in ROS production due to zymosan or C. albicans stimulation. The c-type lectin receptor Dectin-1 can induce the ROS response via activation of Syk kinase with its immunoreceptor tyrosine-based activation motif (ITAM)-like domain upon zymosan or heat-killed C. albicans stimulation. Furthermore, zymosan also activates extracellular signal related kinase ERK1/2 MAPK dependent on Dectin-1. In contrast, Dectin-1 is only moderately involved in activation of ROS and ERK1/2 when stimulated with live C. albicans. Interestingly, activation of Src and Syk kinases is essential to induce the ROS response by live C. albicans. This leads us to conclude that an ITAM-containing receptor or adaptor protein is involved in the recognition of live C. albicans. Using a siRNA-based knock-down assay, we found that one ITAM-containing adaptor protein, DAP12, may contribute to the ROS response upon fungal pathogens such as C. albicans

Microbial stress. From sensing to intracellular and population responses

We initially devised this Research Topic (RT) as a valuable initiative to collect high-quality scientific articles from the participants of the 4th European Federation of Biotechnology (EFB) Microbial Stress meeting held in Kinsale, Ireland, April 2018. The scope of the RT is based on the scientific content of that “Microbial Stress: from Systems to Molecules and back” meeting. Indeed, over 40% of the articles eventually accepted for publication were contributed by meeting participants, but notably the remaining 60% was contributed by authors that work in this field. The collection of 22 original research and 2 review articles, contributed by 163 authors collectively, deal with the many different aspects of the microbial responses to biotic and abiotic stresses, relevant to many fields: from host-pathogen interactions to biotechnology, from bioremediation to food processing, from molecular and single-cell to population studies. The RT showcases the rapid developments of the microbial stress research on a range of microorganisms and stress conditions, and confirms that understanding microbial physiology under stress can be a trigger for the development of new methodologies as well as helping to integrate the knowledge from many different microbiological fields of research. The retrospective analysis of the articles contributed to this RT allowed them to be assigned to one of four main sub-topics: (i) impact of weak organic acids and low pH on micro-organisms, from clinical to biotechnological contexts; (ii) adaptive responses in microbial pathogens to abiotic/environmental stress; (iii) oxidative and metal stress, from clinical to bioremediation contexts, and (iv) regulation of transcription and translation under stress, from epigenetic aspects to the role of second messengers and sRNA