Explore the vast range of titles available.

Fungi, such as Candida albicans , are found in the mammalian gut, but we know little about what they are doing there. Tso et al. put C. albicans under evolutionary pressure by serial passage in mice that were treated with antibiotics and were thus lacking gut bacteria (see the Perspective by d'Enfert). Passage accelerated fungal mutation, especially around the FLO8 gene, resulting in low-virulence phenotypes unable to form hyphae. Nevertheless, these phenotypes stimulated proinflammatory cytokines and conferred transient cross-protection against several other gut inhabitants. However, if an intact microbiota was present, only the virulent hyphal forms persisted. Science , this issue p. 589 ; see also p. 523 Virulence is lost after repeated passage of Candida albicans promotes mutation at yeast-to-hypha transformation genes. Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of “trained immunity.” Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence 1 – 3 . Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin 4 , 5 , which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells 1 , 6 . Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche. Both the yeast and hyphal forms of Candida albicans enable colonization of the mammalian gut, with hyphal cells secreting the toxin candidalysin to inihibit bacteria and support fungal commensalism.

Key Points Candida albicans is a common cause of mucosal infections. In certain groups of immunocompromised patients it also causes life-threatening bloodstream infections that are disseminated to internal organs. It is a polymorphic fungus, being able to grow in yeast, hyphal and pseudohyphal forms. The hyphal form penetrates epithelia and endothelia, causing tissue damage and allowing access to the bloodstream. C. albicans is exquisitely sensitive to the multiple environments that it encounters in the human host and forms hyphae in response to cues such as 37 °C temperature, serum, CO 2 and O 2 tension, and neutral pH. The morphological switch is also regulated by the presence of not only other C. albicans cells but also bacterial cells, both of which are sensed through quorum sensing compounds. Environmental signals are transduced through multiple pathways that target multiple transcription factors, resulting in the expression of a panel of hypha-specific genes. A key pathway is based on cyclic AMP and targets the transcription factor enhanced filamentous growth protein (Efg1). In this pathway, adenylyl cyclase, which is encoded by CYR1 , integrates multiple cues in Ras-dependent and Ras-independent ways. Negative regulation is exerted by the general transcriptional corepressor Tup1, which is targeted to hypha-specific genes by the DNA-binding proteins Nrg1 and Rox1p-like regulator of filamentous growth (Rfg1). The key outputs of the signal transduction pathway are the expression of three genes, UME6 , EED1 and hyphal G1 cyclin protein 1 ( HGC1 ). Overexpression of the transcription factor Ume6 forces ectopic hyphal growth. The role of Eed1 is currently unclear, but current research suggests that it lies upstream of Ume6. Hgc1 is the C. albicans homologue of the S. cerevisiae Ccn1 and Cln2 G1 cyclin pair, which activate the cyclin-dependent kinase cell division control 28 (Cdc28). Hyphae grow in a highly polarized manner from their tip. This requires the delivery of secretory vesicles along actin cables. These vesicles accumulate in a subapical region called the Spitzenkörper before they fuse with the plasma membrane at the tip after docking with a multiprotein structure called the exocyst. Cell separation after cytokinesis is suppressed in hyphae. This suppression involves phosphorylation of Efg1, which then associates with the promoters of genes encoding septum-degrading enzymes, repressing their Ace2-mediated transcription. A second mechanism suppressing cell separation involves the exclusion of the Cdc14 phosphatase from the septin ring, the subunits of which have different dynamic properties in yeast and hyphae. A key role for kinases is emerging in the cell biology of hyphal growth. Hgc1–Cdc28 targets Rga2, Sec2 and Mob2, as well as Efg1. Rga2 is a GTPase-activating protein (GAP) that negatively regulates the GTPase Cdc42, which has a central role in orchestrating polarized growth. Sec2 is the guanosine exchange factor (GEF) that activates the GTPase Sec4, which is required for polarized exocytosis. Mob2 is the activating partner of the kinase Cbk1, which is absolutely required for hyphal growth. Upon hyphal induction, Cdc28 is partnered by a different cyclin, Ccn1, and cooperates with another kinase, growth-inhibitory protein 4 (Gin4), to phosphorylate the septin Cdc11. In response to certain environmental cues, the unicellular budding yeast Candida albicans can also grow as either a pseudohyphal or a hyphal form. In this Review, Sudbery describes the signal transduction pathways and cellular mechanisms that drive polarized hyphal growth and the role of this growth in disease. The fungus Candida albicans is often a benign member of the mucosal flora; however, it commonly causes mucosal disease with substantial morbidity and in vulnerable patients it causes life-threatening bloodstream infections. A striking feature of its biology is its ability to grow in yeast, pseudohyphal and hyphal forms. The hyphal form has an important role in causing disease by invading epithelial cells and causing tissue damage. This Review describes our current understanding of the network of signal transduction pathways that monitors environmental cues to activate a programme of hypha-specific gene transcription, and the molecular processes that drive the highly polarized growth of hyphae.

Polymicrobial biofilms are of large medical importance, but relatively little is known about the role of interspecies interactions for their physiology and virulence. Here, we studied two human pathogens co-occuring in the oral cavity, the opportunistic fungus Candida albicans and the caries-promoting bacterium Streptococcus mutans . Dual-species biofilms reached higher biomass and cell numbers than mono-species biofilms, and the production of extracellular polymeric substances (EPSs) by S. mutans was strongly suppressed, which was confirmed by scanning electron microscopy, gas chromatography–mass spectrometry and transcriptome analysis. To detect interkingdom communication, C. albicans was co-cultivated with a strain of S. mutans carrying a transcriptional fusion between a green fluorescent protein-encoding gene and the promoter for sigX , the alternative sigma factor of S. mutans , which is induced by quorum sensing signals. Strong induction of sigX was observed in dual-species biofilms, but not in single-species biofilms. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion of comS encoding the synthesis of the sigX -inducing peptide precursor abolished this activity, whereas deletion of comC encoding the competence-stimulating peptide precursor had no effect. Transcriptome analysis of S. mutans confirmed induction of comS , sigX , bacteriocins and the downstream late competence genes, including fratricins, in dual-species biofilms. We show here for the first time the stimulation of the complete quorum sensing system of S. mutans by a species from another kingdom, namely the fungus C. albicans , resulting in fundamentally changed virulence properties of the caries pathogen.

Candidalysin is a cytolytic peptide toxin secreted by Candida albicans hyphae and has significantly advanced our understanding of fungal pathogenesis. Candidalysin is critical for mucosal C albicans infections and is known to activate epithelial cells to induce downstream innate immune responses that are associated with protection or immunopathology during oral or vaginal infections. Furthermore, candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. However, the role of candidalysin in driving systemic infections is unknown. In this study, using candidalysin-producing and candidalysin-deficient C albicans strains, we show that candidalysin activates mitogen-activated protein kinase (MAPK) signaling and chemokine secretion in endothelial cells in vitro. Candidalysin induces immune activation and neutrophil recruitment in vivo, and it promotes mortality in zebrafish and murine models of systemic fungal infection. The data demonstrate a key role for candidalysin in neutrophil recruitment and fungal virulence during disseminated systemic C albicans infections.

Candida albicans , the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast–hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo , relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen. Candida albicans is a prominent human fungal pathogen that until now was thought to be an obligate diploid; here it is shown that C. albicans can form viable haploids, that these haploids are able to mate to form heterozygous diploids, and that haploids and their auto-diploids are significantly less fit in vitro and in vivo than heterozygous progenitors or diploids formed by haploid mating pairs. Candida albicans goes halves The common human fungal pathogen Candida albicans has long been considered an obligate diploid organism, with a rare, parasexual tetraploid stage and no meiosis. This has hindered classical genetic studies and made molecular manipulations more difficult than in model yeasts like Saccharomyces cerevisiae . Now Judith Berman and colleagues have identified a viable haploid C. albicans state derived from diploid cells. These cells can be isolated from in vitro stress conditions or following in vivo passage through a mammalian host. Haploids and their auto-diploids are significantly less fit in vivo when compared to heterozygous diploids. The authors have constructed a number of stable haploid strains to facilitate molecular and genetic analyses of C. albicans biology and virulence.

Tolerance to antifungal drug concentrations above the minimal inhibitory concentration (MIC) is rarely quantified, and current clinical recommendations suggest it should be ignored. Here, we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC, and find that it is distinct from susceptibility/resistance. Instead, tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population, and correlates inversely with intracellular drug accumulation. Many adjuvant drugs used in combination with fluconazole, a widely used fungistatic drug, reduce tolerance without affecting resistance. Accordingly, in an invertebrate infection model, adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with low tolerance isolates. Furthermore, isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole. Thus, tolerance correlates with, and may help predict, patient responses to fluconazole therapy. The authors show that antifungal tolerance, defined as the fraction of growth of a fungal pathogen above the minimal inhibitory concentration, is due to the slow growth of subpopulations of cells that overcome drug stress, and that high tolerance is often associated with persistent infections.

Andrew Koh and colleagues report that gut anaerobes in adult mice prevent Candida albicans colonization by inducing an antimicrobial peptide. Candida albicans colonization is required for invasive disease 1 , 2 , 3 . Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization 2 , 4 , but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria—specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes—are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron –induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of \"virulence factors\" important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.