Human Commensal Yeast Research Articles

Metabolic and Phenotypic Changes Induced during N-Acetylglucosamine Signalling in the Fungal Pathogen Candida albicans.

The human commensal yeast Candida albicans is pathogenic and results in a variety of mucosal and deep tissue problems when the host is immunocompromised. Candida exhibits enormous metabolic flexibility and dynamic morphogenetic transition to survive under host niche environmental conditions and to cause virulence. The amino sugar N-acetylglucosamine (GlcNAc) available at the host infection sites, apart from acting as an extremely good carbon and nitrogen source, also induces cellular signalling in this pathogen. In C. albicans, GlcNAc performs multifaceted roles, including GlcNAc scavenging, GlcNAc import and metabolism, morphogenetic transition (yeast-hyphae and white-opaque switch), GlcNAc-induced cell death (GICD), and virulence. Understanding the molecular mechanism(s) involved in GlcNAc-induced cellular processes has become the main focus of many studies. In the current study, we focused on GlcNAc-induced metabolic changes associated with phenotypic changes. Here, we employed gas chromatography-mass spectrometry (GC-MS), which is a high-throughput and sensitive technology, to unveil global metabolomic changes that occur in GlcNAc vs. glucose grown conditions in Candida cells. The morphogenetic transition associated with metabolic changes was analysed by high-resolution field emission scanning electron microscopy (FE-SEM). Metabolite analysis revealed the upregulation of metabolites involved in the glyoxylate pathway, oxidative metabolism, and fatty acid catabolism to probably augment the synthesis of GlcNAc-induced hypha-specific materials. Furthermore, GlcNAc-grown cells showed slightly more sensitivity to amphotericin B treatment. These results all together provide new insights into the development of antifungal therapeutics for the control of candidiasis in humans.

Face/Off: The Interchangeable Side of Candida Albicans.

Due to limited mobility, fungi, like most unicellular organisms, have evolved mechanisms to adapt to sudden chemical and/or physical variation in their environment. Candida albicans is recognized as a model organism to study eukaryotic responses to environmental changes, as this human commensal yeast but also opportunistic pathogen responds to numerous environmental cues through switching morphologies from yeast to hyphae growth. This mechanism is largely controlled by two major pathways: cAMP-PKA and MAPK, but each environmental signal is sensed by specific sensors. However, morphological switching is not the only response C. albicans exerts in response to environmental cues. Recently, fungal cell wall remodeling in response to host-derived environmental cues has been identified as a way for C. albicans to manipulate the innate immune system. The fungal cell wall is composed of a chitin skeleton linked to a network of β-glucan, which anchors proteins and mannans to the fungal cell surface. As localized on the cell surface, these molecules drive interactions with the environment and other cells, particularly with host immune cells. C. albicans is recognized by immune cells such as neutrophils and macrophages via pathogen recognition receptors (PRRs) that bind different components of the cell wall. While β-glucan and mannan are proinflammatory molecules, chitin can induce anti-inflammatory responses. Interestingly, C. albicans is able to regulate the exposure of these pathogen-associated molecular patterns (PAMPs) according to environmental cues resulting in a modulation of the host immune response. This review describes the mechanisms involved in C. albicans response to environmental changes and their effect on immune recognition.

AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans.

The human fungal pathogen Candida albicans is known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex.IMPORTANCECandida albicans is a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans, C. albicans can adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2 levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition in Candida.

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.

Pre-clinical Imaging of Invasive Candidiasis Using ImmunoPET/MR.

The human commensal yeast Candida is the fourth most common cause of hospital-acquired bloodstream infections, with Candida albicans accounting for the majority of the >400,000 life-threatening infections annually. Diagnosis of invasive candidiasis (IC), a disease encompassing candidemia (blood-borne yeast infection) and deep-seated organ infections, is a major challenge since clinical manifestations of the disease are indistinguishable from viral, bacterial and other fungal diseases, and diagnostic tests for biomarkers in the bloodstream such as PCR, ELISA, and pan-fungal β-D-glucan lack either standardization, sensitivity, or specificity. Blood culture remains the gold standard for diagnosis, but test sensitivity is poor and turn-around time slow. Furthermore, cultures can only be obtained when the yeast resides in the bloodstream, with samples recovered from hematogenous infections often yielding negative results. Consequently, there is a pressing need for a diagnostic test that allows the identification of metastatic foci in deep-seated Candida infections, without the need for invasive biopsy. Here, we report the development of a highly specific mouse IgG3 monoclonal antibody (MC3) that binds to a putative β-1,2-mannan epitope present in high molecular weight mannoproteins and phospholipomannans on the surface of yeast and hyphal morphotypes of C. albicans, and its use as a [64Cu]NODAGA-labeled tracer for whole-body pre-clinical imaging of deep-seated C. albicans infections using antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI). When used in a mouse intravenous (i.v.) challenge model that faithfully mimics disseminated C. albicans infections in humans, the [64Cu]NODAGA-MC3 tracer accurately detects infections of the kidney, the principal site of blood-borne candidiasis in this model. Using a strain of the emerging human pathogen Candida auris that reacts with MC3 in vitro, but which is non-infective in i.v. challenged mice, we demonstrate the accuracy of the tracer in diagnosing invasive infections in vivo. This pre-clinical study demonstrates the principle of using antibody-guided molecular imaging for detection of deep organ infections in IC, without the need for invasive tissue biopsy.

Patterns of Human Oral Yeast Species Distribution on Hainan Island in China

Infections by yeast strains of the genus Candida are among the most prevalent fungal infections of humans. These yeasts are common residents of the oral mucosa and other body surfaces. Since most yeast infections are due to endogenous strains and that species of Candida differ in virulence properties and in intrinsic susceptibilities to antifungal drugs, understanding the human commensal yeast flora can help designing effective treatment and prevention strategies against yeast infections. Here, we report the patterns of yeast species distributions in the oral cavities of 1,799 people from Hainan Island in southern China. Based on sequence information at the fungal barcode locus ITS regions, 368 of the 415 obtained oral yeast strains were identified as belonging to 26 yeast species, while the remaining 47 strains all showed significant sequence divergence to the currently described species. The four most common yeast species were C. albicans (42%), C. tropicalis (20%), C. glabrata (5.5%), and C. parapsilosis (4.1%) and 10 of the 26 yeast species were represented by only one strain each. Our analyses identified that the gender of hosts and ethnical background showed no contribution to oral yeast species distributions. However, the health status, place of birth, current residency, and the age of hosts all showed significant contributions to the distributions of the four dominant yeast species. We compared our results with those reported previously and discussed the potential mechanisms for the observed differences in oral yeast species distributions.

Comparative Genomics and the Evolution of Pathogenicity in Human Pathogenic Fungi

Because most fungi have evolved to be free-living in the environment and because the infections they cause are usually opportunistic in nature, it is often difficult to identify specific traits that contribute to fungal pathogenesis. In recent years, there has been a surge in the number of sequenced genomes of human fungal pathogens, and comparison of these sequences has proved to be an excellent resource for exploring commonalities and differences in how these species interact with their hosts. In order to survive in the human body, fungi must be able to adapt to new nutrient sources and environmental stresses. Therefore, genes involved in carbohydrate and amino acid metabolism and transport and genes encoding secondary metabolites tend to be overrepresented in pathogenic species (e.g., Aspergillus fumigatus). However, it is clear that human commensal yeast species such as Candida albicans have also evolved a range of specific factors that facilitate direct interaction with host tissues. The evolution of virulence across the human pathogenic fungi has occurred largely through very similar mechanisms. One of the most important mechanisms is gene duplication and the expansion of gene families, particularly in subtelomeric regions. Unlike the case for prokaryotic pathogens, horizontal transfer of genes between species and other genera does not seem to have played a significant role in the evolution of fungal virulence. New sequencing technologies promise the prospect of even greater numbers of genome sequences, facilitating the sequencing of multiple genomes and transcriptomes within individual species, and will undoubtedly contribute to a deeper insight into fungal pathogenesis.

Oral yeast flora and its ITS sequence diversity among a large cohort of medical students in Hainan, China

The most prevalent fungal infection of humans is candidiasis which is caused by species of Candida that are typical members of the commensal microbial flora of the oral mucosa and other body surfaces. Since species of Candida differ in virulence properties and susceptibilities to anti-fungal drugs, understanding the human commensal yeast flora will have a significant impact on designing treatment and prevention strategies against yeast infections. However, although there is a global interest in Candida species, the global distributions of Candida species remain largely unknown, especially among healthy hosts. Here we report the oral yeast flora from the surveys of over 1,000 medical students in China. Our results showed that this population had a yeast carriage rate (4.5%) much lower than other population samples reported previously from Mainland China (40-70%). In addition, C. albicans was isolated at a much higher frequency than those from other Chinese samples, with a frequency (80.9%) more similar to those in developed regions such as North America. The oral yeast carriage rates and yeast species compositions were similar between male and female students and between the hosts borne and raised on Hainan Island and those borne and raised on Mainland China. Furthermore, the sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster was analyzed for strains of the dominant species, C. albicans. Our analysis identified 14 ITS types among the 41 Hainan isolates of C. albicans. However, only four of the 14 ITS types were identical to those in reference strains from Europe and North America. Taken together, our analyses suggest that the oral yeast flora among host populations in China is highly heterogeneous and that there is a high ITS sequence diversity in the Hainan population of C. albicans.

The human commensal yeast, Candida albicans, has an ancient origin

Candida albicans, the primary causative agent of candidiasis, is a ubiquitous member of the human flora and is capable of causing severe invasive disease. Despite its importance as a human pathogen, little is known concerning those factors creating and maintaining genetic diversity within the species and how extant strains reflect their evolutionary history. Based on nucleotide polymorphism frequencies, we estimated the time to a most recent common ancestor for the species to be about 3–16 million years, with variation due to molecular clock calibration. As C. albicans genotypes have broad geographic associations, this suggests that the origins of DNA sequence variation in extant populations coincided with early hominid evolution. This is consistent with an emerging view of a genetically complex organism that is able to survive under host immunity as an obligate commensal species.

Evidence for aneuploidy and recombination in the human commensal yeast Candida parapsilosis

Isolates of Candida parapsilosis, including representatives of the three major sub-species groups, were screened for single nucleotide polymorphisms (SNPs) by sequencing five independent loci totaling 4 kb per isolate. Group I isolates were highly conserved and in some cases, group I alleles were found in group II and III strains. Unique alleles were also associated with groups II and III, consistent with earlier observations of intergroup divergence. There was no heterozygosity in any strain, and a FACS analysis demonstrated that for all three groups nuclei are variant in size, ranging from 0.5 to 1.0× the size of other diploid yeast genomes. This suggests that natural isolates of C. parapsilosis are aneuploid, with some isolates being essentially haploid. Taken collectively with the observation of group I alleles within group II and III strains, we propose that some form of recombination is occurring between groups.

Towards understanding the evolution of the human commensal yeast Candida albicans.

Allelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in Hardy-Weinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity. Through inference of this and other known attributes of closely related Candida species, such as sequence analysis of IS1 and the ITS2 (internal transcribed spacer 2) region of the rDNA cistron, the deduced phylogeny suggests an evolutionarily recent origin for many frequently isolated strains. This finding will be of interest in the context of understanding pathogenicity and drug resistance in this human commensal yeast.