Candida albicans natural diversity: a resource to dissect fungal commensalism and pathogenesis.

Candida albicans is a ubiquitous fungus of humans. It is not only a component of the oral and intestinal microbiota of most healthy adults but also a major cause of mucosal disorders and life-threatening disseminated infections. Until recently, research on the biology and pathogenesis of the fungus was largely based on a single clinical isolate. We review investigations that have started to dissect a diverse set of C. albicans strains. Using different approaches to leverage the species' phenotypic and/or genetic diversity, these studies illuminate the wide range of interactions between fungus and host. While connecting genetic variants to phenotypes of interest remains challenging, research on C. albicans' natural diversity is central to understand fungal commensalism and pathogenesis.

The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

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 virulence1-3. 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 candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. 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.

RFX transcription factor in the human-associated yeast Candida albicans regulates adhesion to oral epithelium.

Adhesion to mucosal surfaces is a critical step in many bacterial and fungal infections. Here, using a mouse model of oral infection by the human fungal pathobiont Candida albicans, we report the identification of a novel regulator of C. albicans adhesion to the oral mucosa. The regulator is a member of the regulatory factor X (RFX) family of transcription factors, which control cellular processes ranging from genome integrity in model yeasts to tissue differentiation in vertebrates. Mice infected with the C. albicans rfx1 deletion mutant displayed increased fungal burden in tongues compared to animals infected with the reference strain. High-resolution imaging revealed RFX1 transcripts being expressed by C. albicans cells during infection. Concomitant with the increase in fungal burden, the rfx1 mutant elicited an enhanced innate immune response. Transcriptome analyses uncovered HWP1, a gene encoding an adhesion protein that mediates covalent attachment to buccal cells, as a major RFX1-regulated locus. Consistent with this result, we establish that C. albicans adhesion to oral cells is modulated by RFX1 in an HWP1-dependent manner. Our findings expand the repertoire of biological processes controlled by the RFX family and illustrate a mechanism whereby C. albicans can adjust adhesion to the oral epithelium.

Host-derived reactive oxygen species trigger activation of the Candida albicans transcription regulator Rtg1/3.

The signals that denote mammalian host environments and dictate the activation of signaling pathways in human-associated microorganisms are often unknown. The transcription regulator Rtg1/3 in the human fungal pathogen Candida albicans is a crucial determinant of host colonization and pathogenicity. Rtg1/3's activity is controlled, in part, by shuttling the regulator between the cytoplasm and nucleus of the fungus. The host signal(s) that Rtg1/3 respond(s) to, however, have remained unclear. Here we report that neutrophil-derived reactive oxygen species (ROS) direct the subcellular localization of this C. albicans transcription regulator. Upon engulfment of Candida cells by human or mouse neutrophils, the regulator shuttles to the fungal nucleus. Using genetic and chemical approaches to disrupt the neutrophils' oxidative burst, we establish that the oxidants produced by the NOX2 complex-but not the oxidants generated by myeloperoxidase-trigger Rtg1/3's migration to the nucleus. Furthermore, screening a collection of C. albicans kinase deletion mutants, we implicate the MKC1 signaling pathway in the ROS-dependent regulation of Rtg1/3 in this fungus. Finally, we show that Rtg1/3 contributes to C. albicans virulence in the nematode Caenorhabditis elegans in an ROS-dependent manner as the rtg1 and rtg3 mutants display virulence defects in wild-type but not in ROS deficient worms. Our findings establish NOX2-derived ROS as a key signal that directs the activity of the pleiotropic fungal regulator Rtg1/3.

A Fungal Transcription Regulator of Vacuolar Function Modulates Candida albicans Interactions with Host Epithelial Cells.

Microorganisms typically maintain cellular homeostasis despite facing large fluctuations in their surroundings. Microbes that reside on human mucosal surfaces may experience significant variations in nutrient and ion availability as well as pH. Whether the mechanisms employed by these microbial cells to sustain homeostasis directly impact on the interplay with the host's mucosae remains unclear. Here, we report that the previously uncharacterized transcription regulator ZCF8 in the human-associated yeast Candida albicans maintains vacuole homeostasis when the fungus faces fluctuations in nitrogen. Genome-wide identification of genes directly regulated by Zcf8p followed by fluorescence microscopy to define their subcellular localization uncovered the fungal vacuole as a top target of Zcf8p regulation. Deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and luminal pH and rendered the fungus resistant or susceptible to nigericin and brefeldin A, two drugs that impair vacuole and associated functions. Furthermore, we establish that the regulator modulates C. albicans attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Our findings, therefore, suggest that fungal vacuole physiology regulation is intrinsically linked to, and shapes to a significant extent, the physical interactions that Candida cells establish with mammalian mucosal surfaces. IMPORTANCE Candida albicans is a fungus that resides on various human mucosal surfaces. Individuals with debilitated immune systems are prone to develop C. albicans infections, which can range in severity from mucosal disease (e.g., oral thrush in AIDS patients) to life-threatening conditions (e.g., deep-seated, disseminated infections in patients undergoing organ transplants). Understanding the cellular and molecular mechanisms that this eukaryotic microbe employs to colonize different parts of the human body and to cause disease will lay the foundation for the development of novel strategies for preventing and treating C. albicans infections. This report establishes the fungal vacuole, a key organelle to the overall fungal physiology, as a key determinant of the interplay between C. albicans and mammalian mucosal surfaces.

The interplay between gut bacteria and the yeast Candida albicans.

The fungus Candida albicans is a ubiquitous member of the human gut microbiota. Hundreds or thousands of bacterial taxa reside together with this fungus in the intestine, creating a milieu with myriad opportunities for inter-kingdom interactions. Indeed, recent studies examining the broader composition - that is, monitoring not only bacteria but also the often neglected fungal component - of the gut microbiota hint that there are significant interdependencies between fungi and bacteria. Gut bacteria closely associate with C. albicans cells in the colon, break down and feed on complex sugars decorating the fungal cell wall, and shape the intestinal microhabitats occupied by the fungus. Peptidoglycan subunits released by bacteria upon antibiotic treatment can promote C. albicans dissemination from the intestine, seeding bloodstream infections that often become life-threatening. Elucidating the principles that govern the fungus-bacteria interplay may open the door to novel approaches to prevent C. albicans infections originating in the gut.

Imaging and Quantification of mRNA Molecules at Single-Cell Resolution in the Human Fungal Pathogen Candida albicans.

The study of gene expression in fungi has typically relied on measuring transcripts in populations of cells. A major disadvantage of this approach is that the transcripts' spatial distribution and stochastic variation among individual cells within a clonal population is lost. Traditional fluorescence in situ hybridization techniques have been of limited use in fungi due to poor specificity and high background signal. Here, we report that in situ hybridization chain reaction (HCR), a method that employs split-initiator probes to trigger signal amplification upon mRNA-probe hybridization, is ideally suited for the imaging and quantification of low-abundance transcripts at single-cell resolution in the fungus Candida albicans. We show that HCR allows the absolute quantification of transcripts within a cell by microscopy as well as their relative quantification by flow cytometry. mRNA imaging also revealed the subcellular localization of specific transcripts. Furthermore, we establish that HCR is amenable to multiplexing by visualizing different transcripts in the same cell. Finally, we combine HCR with immunostaining to image specific mRNAs and proteins simultaneously within a single C. albicans cell. The fungus is a major pathogen in humans where it can colonize and invade mucosal surfaces and most internal organs. The technical development that we introduce, therefore, paves the way to study the patterns of expression of pathogenesis-associated C. albicans genes in infected organs at single-cell resolution. IMPORTANCE Tools to visualize and quantify transcripts at single-cell resolution have enabled the dissection of spatiotemporal patterns of gene expression in animal cells and tissues. Yet the accurate quantification of transcripts at single-cell resolution remains challenging for the much smaller microbial cells. Widespread phenomena such as stochastic variation in transcript levels among cells-even within a clonal population-seem to play important roles in the biology of many microorganisms. Investigating this process requires microbial cell-optimized procedures to image and measure mRNAs at single-molecule resolution. In this report, we adapt and expand in situ hybridization chain reaction (HCR) combined with split-initiator probes to visualize transcripts in the human-pathogenic fungus Candida albicans at high resolution.

Fungi of the human gut microbiota: Roles and significance.

It is becoming increasingly clear that fungi are important components of the gut microbiota. Fungi residing in the human intestine, for example, elicit the induction of T helper 17 cells, which are central orchestrators of protective immune responses. Likewise, fungal members of the intestinal microbiota have been shown to influence the immunological responses of the mammalian host by dampening or promoting local inflammatory responses. Here I review some of the latest developments regarding symbiotic fungi of the gastrointestinal tract and the consequences that fungal dysbiosis may have on human health. A major focus of the review is on the relationship between Candida albicans, the most prominent fungus inhabiting the human gut, and the mammalian host. Advances in the field underscore the need to further investigate the fungi that inhabit the human body to understand how the mixed array of microbes that constitute our microbiota contribute to health and disease.

Gut Bacteria Shape Intestinal Microhabitats Occupied by the Fungus Candida albicans.

The human gut microbiota is composed of diverse microbes that not only compete but also rely on one another for resources and access to microhabitats in the intestine [1, 2]. Indeed, recent efforts to map the microbial biogeography of the gastrointestinal tract have revealed positive and negative co-associations between bacterial taxa [3, 4]. Here, we examine the spatial organization that the most prominent fungus of the human flora, Candida albicans, adopts in the gut of gnotobiotic mice either as the sole colonizer or in the presence of single bacterial species. We observe that, as a lone colonizer, C. albicans cells are distributed either adjacent to the inner mucus layer in the colon or throughout the intestinal lumen. In contrast to this pattern, in the presence of the saccharolytic Bacteroides thetaiotaomicron, the fungal cells localize to the interior of a Bacteroides-promoted outer mucus layer in which fungal and bacterial cells are in close association. We show that, in vitro, although mucin provides minimal support to the proliferation of the fungus, barely altering its transcriptional landscape, Bacteroides- and glucanase-processed mucin can better fuel the growth of C. albicans. Our observations illustrate how a commensal fungus can settle in an intestinal microhabitat generated by the presence of a single gut bacterial taxon.

The Regulatory Proteins Rtg1/3 Govern Sphingolipid Homeostasis in the Human-Associated Yeast Candida albicans.

Integrating nutrient sensing with the synthesis of complex molecules is a central feature of metabolism. Yet the regulatory mechanisms underlying such integration are often unknown. Here, we establish that the transcription regulators Rtg1/3 are key determinants of sphingolipid homeostasis in the human fungal pathogen Candida albicans. Quantitative analysis of the C. albicans lipidome reveals Rtg1/3-dependent alterations in all complex sphingolipids and their precursors, ceramides. Mutations in the regulators render the fungus susceptible to myriocin, a sphingolipid synthesis inhibitor. Rtg1/3 exert control on the expression of several enzymes involved in the synthesis of sphingolipids' building blocks, and the regulators are activated upon engulfment of C. albicans cells by human neutrophils. We demonstrate that Rtg1p and Rtg3p are regulated at two levels, one in response to sphingolipids and the other by the nutrient sensor TOR. Our findings, therefore, indicate that the Rtg1/3 system integrates nutrient sensing into the synthesis of complex lipids.

Remasking of Candida albicans ß-Glucan in Response to Environmental pH Is Regulated by Quorum Sensing.

Candida albicans is a commensal yeast of the human gut which is tolerated by the immune system but has the potential to become an opportunistic pathogen. One way in which C. albicans achieves this duality is through concealing or exposing cell wall pathogen-associated molecular patterns (PAMPs) in response to host-derived environment cues (pH, hypoxia, and lactate). This cell wall remodeling allows C. albicans to evade or hyperactivate the host's innate immune responses, leading to disease. Previously, we showed that adaptation of C. albicans to acidic environments, conditions encountered during colonization of the female reproductive tract, induces significant cell wall remodeling resulting in the exposure of two key fungal PAMPs (ß-glucan and chitin). Here, we report that this pH-dependent cell wall remodeling is time dependent, with the initial change in pH driving cell wall unmasking, which is then remasked at later time points. Remasking of ß-glucan was mediated via the cell density-dependent fungal quorum sensing molecule farnesol, while chitin remasking was mediated via a small, heat-stable, nonproteinaceous secreted molecule(s). Transcript profiling identified a core set of 42 genes significantly regulated by pH over time and identified the transcription factor Efg1 as a regulator of chitin exposure through regulation of CHT2 This dynamic cell wall remodeling influenced innate immune recognition of C. albicans, suggesting that during infection, C. albicans can manipulate the host innate immune responses.IMPORTANCECandida albicans is part of the microbiota of the skin and gastrointestinal and reproductive tracts of humans and has coevolved with us for millennia. During that period, C. albicans has developed strategies to modulate the host's innate immune responses, by regulating the exposure of key epitopes on the fungal cell surface. Here, we report that exposing C. albicans to an acidic environment, similar to the one of the stomach or vagina, increases the detection of the yeast by macrophages. However, this effect is transitory, as C. albicans is able to remask these epitopes (glucan and chitin). We found that glucan remasking is controlled by the production of farnesol, a molecule secreted by C. albicans in response to high cell densities. However, chitin-remasking mechanisms remain to be identified. By understanding the relationship between environmental sensing and modulation of the host-pathogen interaction, new opportunities for the development of innovative antifungal strategies are possible.

mSphere of Influence: a Systematic Approach To Dissect Virulence Traits in Candida albicans.

J. Christian Pérez studies the interplay between Candida albicans and the mammalian host. In this mSphere of Influence article, he reflects on how "Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity" (S. M. Noble, S. French, L. A. Kohn, V. Chen, et al., Nat Genet 42:590-598, 2010, https://doi.org/10.1038/ng.605) provided tools and a blueprint for open-ended genetic screens in an organism that had been a challenge for genetic manipulation.

Candida albicans dwelling in the mammalian gut.

The yeast Candida albicans inhabits the gastrointestinal tract of most healthy adults, seemingly living there as a harmless commensal. The fungus on occasion disseminates from the gut to other internal organs causing life-threatening infections. Here, I review some of the most exciting advances in the study of gut colonization by C. albicans in the last few years. These developments highlight the close interplay between C. albicans and cohabiting microbes, the responses that commensal fungi elicit from the mammalian host, and the genetic determinants that allow the fungus to thrive in such a crowded and demanding ecosystem.

Identification of Candida albicans regulatory genes governing mucosal infection.

The fungus Candida albicans thrives on a variety of human mucosae, yet the fungal determinants that contribute to fitness on these surfaces remain underexplored. Here, by screening a collection of C. albicans deletion strains in a mouse model of oral infection (oropharyngeal candidiasis), we identify several novel regulatory genes that modulate the fitness of the fungus in this locale. We investigate in detail the interplay between the host mucosa and one of the identified mutants and establish that the C. albicans transcription regulator CUP9 is a key determinant of mucosal colonisation. Deletion of cup9 resulted in the formation of more foci of colonisation and heightened persistence in infected tongues. Furthermore, the cup9 mutant produced longer and denser filaments in the oral mucosa without eliciting an enhanced local immune response. Consistent with its role in oral colonisation, we show that CUP9's top target of regulation is a major effector of Candida's adherence to buccal cells. Finally, we establish that CUP9 also governs the interplay of the fungus with vaginal epithelial cells and has a role in vaginal infections, another common mucosal disease associated with Candida. Thus, our findings reveal a mechanism whereby C. albicans can regulate proliferation on mucosal surfaces.

Diversification of DNA binding specificities enabled SREBP transcription regulators to expand the repertoire of cellular functions that they govern in fungi.

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus' natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators.

The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice.

Many microorganisms that cause systemic, life-threatening infections in humans reside as harmless commensals in our digestive tract. Yet little is known about the biology of these microbes in the gut. Here, we visualize the interface between the human commensal and pathogenic fungus Candida albicans and the intestine of mice, a surrogate host. Because the indigenous mouse microbiota restricts C. albicans settlement, we compared the patterns of colonization in the gut of germ free and antibiotic-treated conventionally raised mice. In contrast to the heterogeneous morphologies found in the latter, we establish that in germ free animals the fungus almost uniformly adopts the yeast cell form, a proxy of its commensal state. By screening a collection of C. albicans transcription regulator deletion mutants in gnotobiotic mice, we identify several genes previously unknown to contribute to in vivo fitness. We investigate three of these regulators-ZCF8, ZFU2 and TRY4-and show that indeed they favor the yeast form over other morphologies. Consistent with this finding, we demonstrate that genetically inducing non-yeast cell morphologies is detrimental to the fitness of C. albicans in the gut. Furthermore, the identified regulators promote adherence of the fungus to a surface covered with mucin and to mucus-producing intestinal epithelial cells. In agreement with this result, histology sections indicate that C. albicans dwells in the murine gut in close proximity to the mucus layer. Thus, our findings reveal a set of regulators that endows C. albicans with the ability to endure in the intestine through multiple mechanisms.

A Candida albicans regulator of disseminated infection operates primarily as a repressor and governs cell surface remodeling.

Virulence traits are often controlled by transcription regulators, i.e. sequence-specific DNA-binding proteins. The regulators that sustain microbial proliferation in the host typically work by promoting the expression of the genes that mediate such traits. Here, we report a singular example in the human fungal pathogen Candida albicans in which a transcription regulator functions by repressing the expression of virulence genes, yet its overall role is to promote virulence. We explain this apparent paradox by establishing that a major function of this protein, Zcf21p, is to set a default state of low expression of multiple cell wall components which include virulence determinants. These components comprise GPI-anchored proteins, adhesins and enzymes that synthesize cell wall sugar decorations. Deletion or overexpression of ZCF21 results in cell wall structure modifications that influence recognition and elimination of the fungus by macrophages. By leveling off the expression of adhesins, ZCF21 also prevents C. albicans self-aggregation. Balancing the expression of cell wall components - virulence determinants included - is, therefore, critical for C. albicans to assemble a cell surface configuration that is suitable to colonize mammalian tissues and evade immune surveillance.

Reshuffling transcriptional circuits: how microorganisms adapt to colonize the human body.

Several hundred taxa of microorganisms-including bacteria, archaea and eukaryotes-inhabit the human body. What did it take for these species to become stable residents of humans? Recent reports illustrate how evolutionary changes in transcriptional circuits played a pivotal role in the adaptation of single-celled eukaryotes to colonize mammals.

How duplicated transcription regulators can diversify to govern the expression of nonoverlapping sets of genes.

The duplication of transcription regulators can elicit major regulatory network rearrangements over evolutionary timescales. However, few examples of duplications resulting in gene network expansions are understood in molecular detail. Here we show that four Candida albicans transcription regulators that arose by successive duplications have differentiated from one another by acquiring different intrinsic DNA-binding specificities, different preferences for half-site spacing, and different associations with cofactors. The combination of these three mechanisms resulted in each of the four regulators controlling a distinct set of target genes, which likely contributed to the adaption of this fungus to its human host. Our results illustrate how successive duplications and diversification of an ancestral transcription regulator can underlie major changes in an organism's regulatory circuitry.

Candida albicans commensalism and pathogenicity are intertwined traits directed by a tightly knit transcriptional regulatory circuit.

Systemic, life-threatening infections in humans are often caused by bacterial or fungal species that normally inhabit a different locale in our body, particularly mucosal surfaces. A hallmark of these opportunistic pathogens, therefore, is their ability to thrive in disparate niches within the host. In this work, we investigate the transcriptional circuitry and gene repertoire that enable the human opportunistic fungal pathogen Candida albicans to proliferate in two different niches. By screening a library of transcription regulator deletion strains in mouse models of intestinal colonization and systemic infection, we identified eight transcription regulators that play roles in at least one of these models. Using genome-wide chromatin immunoprecipitation, we uncovered a network comprising ∼800 target genes and a tightly knit transcriptional regulatory circuit at its core. The network is enriched with genes upregulated in C. albicans cells growing in the host. Our findings indicate that many aspects of commensalism and pathogenicity are intertwined and that the ability of this microorganism to colonize multiple niches relies on a large, integrated circuit.