Combatting Fungal Infections: Advances in Antifungal Polymeric Nanomaterials.

Fungal pathogens cause over 6.5 million life-threatening systemic infections annually, with mortality rates ranging from 20 to 95%, even with medical intervention. The World Health Organization has recently emphasized the urgent need for new antifungal drugs. However, the range of effective antifungal agents remains limited and resistance is increasing. This Review explores the current landscape of fungal infections and antifungal drugs, focusing on synthetic polymeric nanomaterials like nanoparticles that enhance the physicochemical properties of existing drugs. Additionally, we examine intrinsically antifungal polymers that mimic naturally occurring peptides. Advances in polymer characterization and synthesis now allow precise design and screening for antifungal activity, biocompatibility, and drug interactions. These antifungal polymers represent a promising new class of drugs for combating fungal infections.

A synthetic peptide mimic kills Candida albicans and synergistically prevents infection.

More than two million people worldwide are affected by life-threatening, invasive fungal infections annually. Candida species are the most common cause of nosocomial, invasive fungal infections and are associated with mortality rates above 40%. Despite the increasing incidence of drug-resistance, the development of novel antifungal formulations has been limited. Here we investigate the antifungal mode of action and therapeutic potential of positively charged, synthetic peptide mimics to combat Candida albicans infections. Our data indicates that these synthetic polymers cause endoplasmic reticulum stress and affect protein glycosylation, a mode of action distinct from currently approved antifungal drugs. The most promising polymer composition damaged the mannan layer of the cell wall, with additional membrane-disrupting activity. The synergistic combination of the polymer with caspofungin prevented infection of human epithelial cells in vitro, improved fungal clearance by human macrophages, and significantly increased host survival in a Galleria mellonella model of systemic candidiasis. Additionally, prolonged exposure of C. albicans to the synergistic combination of polymer and caspofungin did not lead to the evolution of tolerant strains in vitro. Together, this work highlights the enormous potential of these synthetic peptide mimics to be used as novel antifungal formulations as well as adjunctive antifungal therapy.

Bacteria-derived short-chain fatty acids as potential regulators of fungal commensalism and pathogenesis.

The human gastrointestinal microbiome encompasses bacteria, fungi, and viruses forming complex bionetworks which, for organismal health, must be in a state of homeostasis. An important homeostatic mechanism derives from microbial competition, which maintains the relative abundance of microbial species in a healthy balance. Microbes compete for nutrients and secrete metabolites that inhibit other microbes. Short-chain fatty acids (SCFAs) are one such class of metabolites made by gut bacteria to very high levels. SCFAs are metabolised by microbes and host cells and have multiple roles in regulating cell physiology. Here, we review the mechanisms by which SCFAs regulate the fungal gut commensal Candida albicans. We discuss SCFA's ability to inhibit fungal growth, limit invasive behaviours and modulate cell surface antigens recognised by immune cells. We review the mechanisms underlying these roles: regulation of gene expression, metabolism, signalling and SCFA-driven post-translational protein modifications by acylation, which contribute to changes in acylome dynamics of C. albicans with potentially large consequences for cell physiology. Given that the gut mycobiome is a reservoir for systemic disease and has also been implicated in inflammatory bowel disease, understanding the mechanisms by which bacterial metabolites, such as SCFAs, control the mycobiome might provide therapeutic avenues.

Mimicking Charged Host-Defense Peptides to Tune the Antifungal Activity and Biocompatibility of Amphiphilic Polymers.

Invasive fungal infections impose a substantial global health burden. They cause more than 1.5 million deaths annually and are insufficiently met by the currently approved antifungal drugs. Antifungal peptides are a promising alternative to existing antifungal drugs; however, they can be challenging to synthesize, and are often susceptible to proteases in vivo. Synthetic polymers which mimic the properties of natural antifungal peptides can circumvent these limitations. In this study, we developed a library of 29 amphiphilic polyacrylamides with different charged units, namely, amines, guanidinium, imidazole, and carboxylic acid groups, representative of the natural amino acids lysine, arginine, histidine, and glutamic acid. Ternary polymers incorporating primary ammonium (lysine-like) or imidazole (histidine-like) groups demonstrated superior activity against Candida albicans and biocompatibility with mammalian cells compared to the polymers containing the other charged groups. Furthermore, a combination of primary ammonium, imidazole, and guanidinium (arginine-like) within the same polymer outperformed the antifungal drug amphotericin B in terms of therapeutic index and exhibited fast C. albicans-killing activity. The most promising polymer compositions showed synergistic effects in combination with caspofungin and fluconazole against C. albicans and additionally demonstrated activity against other clinically relevant fungi. Collectively, these results indicate the strong potential of these easily producible polymers to be used as antifungals.

Effect of Star Topology Versus Linear Polymers on Antifungal Activity and Mammalian Cell Toxicity.

The global increase in invasive fungal infections and the emergence of drug-resistant strains demand the urgent development of novel antifungal drugs. In this context, synthetic polymers with diverse compositions, mimicking natural antimicrobial peptides, have shown promising potential for combating fungal infections. This study investigates how altering polymer end-groups and topology from linear to branched star-like structures affects their efficacy against Candida spp., including clinical isolates. Additionally, the polymers' biocompatibility is accessed with murine embryonic fibroblasts and red blood cells in vitro. Notably, a low-molecular weight star polymer outperforms both its linear polymeric counterparts and amphotericin B (AmpB) in terms of an improved therapeutic index and reduced haemolytic activity, despite a higher minimum inhibitory concentration against Candida albicans (C. albicans) SC5314 (16-32 µg mL-1 vs 1 µg mL-1 for AmpB). These findings demonstrate the potential of synthetic polymers with diverse topologies as promising candidates for antifungal applications.

Architecture of the dynamic fungal cell wall.

The fungal cell wall is essential for growth and survival, and is a key target for antifungal drugs and the immune system. The cell wall must be robust but flexible, protective and shielding yet porous to nutrients and membrane vesicles and receptive to exogenous signals. Most fungi have a common inner wall skeleton of chitin and ß-glucans that functions as a flexible viscoelastic frame to which a more diverse set of outer cell wall polymers and glycosylated proteins are attached. Whereas the inner wall largely determines shape and strength, the outer wall confers properties of hydrophobicity, adhesiveness, and chemical and immunological heterogeneity. The spatial organization and dynamic regulation of the wall in response to prevailing growth conditions enable fungi to thrive within changing, diverse and often hostile environments. Understanding this architecture provides opportunities to develop diagnostics and drugs to combat life-threatening fungal infections.

Human gut bifidobacteria inhibit the growth of the opportunistic fungal pathogen Candida albicans.

The human gut microbiota protects the host from invading pathogens and the overgrowth of indigenous opportunistic species via a process called colonization resistance. Here, we investigated the antagonistic activity of human gut bacteria towards Candida albicans, an opportunistic fungal pathogen that can cause severe infections in susceptible individuals. Coculture batch incubations of C. albicans in the presence of faecal microbiota from six healthy individuals revealed varying levels of inhibitory activity against C. albicans. 16S rRNA gene amplicon profiling of these faecal coculture bacterial communities showed that the Bifidobacteriaceae family, and Bifidobacterium adolescentis in particular, were most correlated with antagonistic activity against C. albicans. Follow-up mechanistic studies performed under anaerobic conditions confirmed that culture supernatants of Bifidobacterium species, particularly B. adolescentis, inhibited C. albicans in vitro. Fermentation acids (FA), including acetate and lactate, present in the bifidobacterial supernatants were important contributors to inhibitory activity. However, increasing the pH of both bacterial supernatants and mixtures of FA reduced their anti-Candida effects, indicating a combinatorial effect of prevailing pH and FA. This work, therefore, demonstrates potential mechanisms underpinning gut microbiome-mediated colonization resistance against C. albicans, and identifies particularly inhibitory components such as bifidobacteria and FA as targets for further study.

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.

Rational Design of an Antifungal Polyacrylamide Library with Reduced Host-Cell Toxicity.

Life-threatening invasive fungal infections represent an urgent threat to human health worldwide. The limited set of antifungal drugs has critical constraints such as resistance development and/or adverse side effects. One approach to overcome these limitations is to mimic naturally occurring antifungal peptides called defensins. Inspired by their advantageous amphiphilic properties, a library of 35 synthetic, linear, ternary polyacrylamides was prepared by controlled/living radical polymerization. The effect of the degree of polymerization (20, 40, and 100) and varying hydrophobic functionalities (branched, linear, cyclic, or aromatic differing in their number of carbons) on their antifungal activity was investigated. Short copolymers with a calculated log P of ∼1.5 revealed optimal activity against the major human fungal pathogen Candida albicans and other pathogenic fungal species with limited toxicity to mammalian host cells (red blood cells and fibroblasts). Remarkably, selected copolymers outperformed the commercial antifungal drug amphotericin B, with respect to the therapeutic index, highlighting their potential as novel antifungal compounds.

Scalar nanostructure of the Candida albicans cell wall; a molecular, cellular and ultrastructural analysis and interpretation.

Despite the importance of fungal cell walls as the principle determinant of fungal morphology and the defining element determining fungal interactions with other cells, few scalar models have been developed that reconcile chemical and microscopic attributes of its structure. The cell wall of the fungal pathogen Candida albicans is comprised of an amorphous inner skeletal layer of ß(1,3)- and ß(1,6)-glucan and chitin and an outer fibrillar layer thought to be dominated by highly mannosylated cell wall proteins. The architecture of these two layers can be resolved at the electron microscopy level, but the visualised structure of the wall has not yet been defined precisely in chemical terms. We have therefore examined the precise structure, location and molecular sizes of the cell wall components using transmission electron microscopy and tomography and tested predictions of the cell wall models using mutants and agents that perturb the normal cell wall structure. We demonstrate that the fibrils are comprised of a frond of N-linked outer chain mannans linked to a basal layer of GPI-proteins concentrated in the mid-wall region and that the non-elastic chitin microfibrils are cantilevered with sufficient lengths of non-fibrillar chitin and/or ß-glucan to enable the chitin-glucan cage to flex, e.g. during morphogenesis and osmotic swelling. We present the first three-dimensional nano-scalar model of the C. albicans cell wall which can be used to test hypotheses relating to the structure-function relationships that underpin the pathobiology of this fungal pathogen.

The Viscoelastic Properties of the Fungal Cell Wall Allow Traffic of AmBisome as Intact Liposome Vesicles.

The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles.IMPORTANCE AmBisome is a broad-spectrum fungicidal antifungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the fungal cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity. This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the cell membrane without unloading the polyene in the cell wall. In addition, these findings suggest that extracellular vesicles may also be able to pass through the cell wall to deliver soluble and membrane-bound effectors and other molecules to the extracellular space.

Adaptation of Candida albicans to environmental pH induces cell wall remodelling and enhances innate immune recognition.

Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and ß-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory ß-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased ß-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this "unmasking" of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection.

The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence.

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.

Cell wall protection by the Candida albicans class I chitin synthases.

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.

Fungal chitin dampens inflammation through IL-10 induction mediated by NOD2 and TLR9 activation.

Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.

The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans.

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.

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.

A systems biology analysis of long and short-term memories of osmotic stress adaptation in fungi.

BACKGROUND: Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans. RESULTS: We combined modeling and experimental approaches to study osmotic stress responses in S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments. CONCLUSIONS: Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts.

Combinatorial stresses kill pathogenic Candida species.

Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.

Rapid detection of aneuploidy following the generation of mutants in Candida albicans.

Techniques used to generate mutants in Candida albicans commonly result in additional and undesired genetic rearrangements. Detection of aneuploidy is, therefore, an important step forward in the quality control of mutant phenotypes. In this chapter, we describe how to extract genomic DNA and perform a quantitative multiplex PCR to compare the karyotype of any mutant strain to that of its parent and allow the detection of any unwanted aneuploidy.